Anti-FcRH5 antibodies and immunoconjugates and methods of use

ABSTRACT

A method of determining the presence of FcRH5 in a biological sample suspected of containing FcRH5 is provided comprising exposing the biological sample to an isolated monoclonal anti-FcRH5 antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/751,791, and claims priority under Section 119(e) and the benefit ofU.S. Provisional Application Ser. Nos. 61/211,754 filed Apr. 1, 2009 and61/166,214 filed Apr. 2, 2009, the entire disclosures of which areincorporated herein by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Mar. 25, 2010, is namedGNE0351R.txt, and is 89,184 bytes in size.

FIELD OF THE INVENTION

The present invention is directed to compositions of matter useful forthe treatment of hematopoietic tumor in mammals and to methods of usingthose compositions of matter for the same.

BACKGROUND OF THE INVENTION

Malignant tumors (cancers) are the second leading cause of death in theUnited States, after heart disease (Boring et al., CA Cancel J. Clin.43:7 (1993)). Cancer is characterized by the increase in the number ofabnormal, or neoplastic, cells derived from a normal tissue whichproliferate to form a tumor mass, the invasion of adjacent tissues bythese neoplastic tumor cells, and the generation of malignant cellswhich eventually spread via the blood or lymphatic system to regionallymph nodes and to distant sites via a process called metastasis. In acancerous state, a cell proliferates under conditions in which normalcells would not grow. Cancer manifests itself in a wide variety offorms, characterized by different degrees of invasiveness andaggressiveness.

Cancers which involve cells generated during hematopoiesis, a process bywhich cellular elements of blood, such as lymphocytes, leukocytes,platelets, erythrocytes and natural killer cells are generated arereferred to as hematopoietic cancers. Lymphocytes which can be found inblood and lymphatic tissue and are critical for immune response arecategorized into two main classes of lymphocytes: B lymphocytes (Bcells) and T lymphocytes (T cells), which mediate humoral and cellmediated immunity, respectively.

B cells mature within the bone marrow and leave the marrow expressing anantigen-binding antibody on their cell surface. When a naive B cellfirst encounters the antigen for which its membrane-bound antibody isspecific, the cell begins to divide rapidly and its progenydifferentiate into memory B cells and effector cells called “plasmacells”. Memory B cells have a longer life span and continue to expressmembrane-bound antibody with the same specificity as the original parentcell. Plasma cells do not produce membrane-bound antibody but insteadproduce the antibody in a form that can be secreted. Secreted antibodiesare the major effector molecule of humoral immunity.

T cells mature within the thymus which provides an environment for theproliferation and differentiation of immature T cells. During T cellmaturation, the T cells undergo the gene rearrangements that produce theT-cell receptor and the positive and negative selection which helpsdetermine the cell-surface phenotype of the mature T cell.Characteristic cell surface markers of mature T cells are the CD3:T-cellreceptor complex and one of the coreceptors, CD4 or CD8.

In attempts to discover effective cellular targets for cancer therapy,researchers have sought to identify transmembrane or otherwisemembrane-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchmembrane-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies. In this regard, it is noted that antibody-based therapy hasproved very effective in the treatment of certain cancers. For example,HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco,Calif.) are antibodies that have been used successfully to treat breastcancer and non-Hodgkin's lymphoma, respectively. More specifically,HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibodythat selectively binds to the extracellular domain of the humanepidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 proteinoverexpression is observed in 25-30% of primary breast cancers. RITUXAN®is a genetically engineered chimeric murine/human monoclonal antibodydirected against the CD20 antigen found on the surface of normal andmalignant B lymphocytes. Both these antibodies are recombinantlyproduced in CHO cells.

In other attempts to discover effective cellular targets for cancertherapy, researchers have sought to identify (1) non-membrane-associatedpolypeptides that are specifically produced by one or more particulartype(s) of cancer cell(s) as compared to by one or more particulartype(s) of non-cancerous normal cell(s), (2) polypeptides that areproduced by cancer cells at an expression level that is significantlyhigher than that of one or more normal non-cancerous cell(s), or (3)polypeptides whose expression is specifically limited to only a single(or very limited number of different) tissue type(s) in both thecancerous and non-cancerous state (e.g., normal prostate and prostatetumor tissue). Such polypeptides may remain intracellularly located ormay be secreted by the cancer cell. Moreover, such polypeptides may beexpressed not by the cancer cell itself, but rather by cells whichproduce and/or secrete polypeptides having a potentiating orgrowth-enhancing effect on cancer cells. Such secreted polypeptides areoften proteins that provide cancer cells with a growth advantage overnormal cells and include such things as, for example, angiogenicfactors, cellular adhesion factors, growth factors, and the like.Identification of antagonists of such non-membrane associatedpolypeptides would be expected to serve as effective therapeutic agentsfor the treatment of such cancers. Furthermore, identification of theexpression pattern of such polypeptides would be useful for thediagnosis of particular cancers in mammals.

Despite the above identified advances in mammalian cancer therapy, thereis a great need for additional therapeutic agents capable of detectingthe presence of tumor in a mammal and for effectively inhibitingneoplastic cell growth, respectively. Accordingly, it is an objective ofthe present invention to identify polypeptides, cellmembrane-associated, secreted or intracellular polypeptides whoseexpression is specifically limited to only a single (or very limitednumber of different) tissue type(s), hematopoietic tissues, in both acancerous and non-cancerous state, and to use those polypeptides, andtheir encoding nucleic acids, to produce compositions of matter usefulin the therapeutic treatment and/or detection of hematopoietic cancer inmammals.

Abnormalities of chromosome 1q21 are common in B cell malignancies,including B cell lymphoma and myeloma, but the genes targeted by theseaberrations are largely unknown. Chromosomal abnormalities involvingband 1q21-q23 are among the most frequent genetic lesions in both B cellnon-Hodgkins lymphoma and multiple myeloma. Among non-Hodgkin's lymphomasubtypes, translocation breakpoints at 1q21-q23, includingtranslocations and duplications, have been reported, often as the singlechromosomal abnormality in follicular and diffuse large B cell lymphoma(DLCL) in marginal-zone B cell lymphoma and in Burkitt lymphoma. Bycloning the breakpoints of a t(1:14)(q21:q32) chromosomal translocationin a myeloma cell line, two genes were identified, termed immunoglobulinsuperfamily receptor translocation associated (IRTA) 1 and IRTA2. IRTA2is identical to sequences identified as BXMAS1 (Nakayama et al., Biochm.Biophys. Res. Commun. 285:830-7, 2001) and FcRH5 (Davis et al., Proc.Natl. Acad. Sci. USA 98:9772-7, 2001). Both IRTA1 and IRTA2 are membersof a family of related genes, the IRTA.

FcRH5 (or IRTA2) is a cell surface receptor with homologies to the Fcreceptor families. It is normally expressed in mature B cells, and has adifferent distribution in peripheral lymphoid organs than FcRH4 (IRTA1).IRTA1 is expressed in marginal zone B cells, while IRTA2 is alsoexpressed in germinal center centrocytes and in immunoblasts. IRTA2expression is deregulated in multiple myeloma and Burkitt lymphoma celllines with 1q21 abnormalities (see Miller et al., Blood 99:2662-2669,2002). The high frequency of involvement of 1q21 structuralrearrangements in B cell malignancies suggests that IRTA1 and IRTA2 arecritical to the pathogenesis of these diseases (see published PCTApplication No. WO 01/38490; U.S. Published Patent Application No.20080292632) (see also Polson, et al. Int Immunol. 2006 September;18(9):1363-73).

Given the expression of FcRH5, it is beneficial to produce therapeuticantibodies to the FcRH5 antigen that create minimal or no antigenicitywhen administered to patients, especially for chronic treatment. Thepresent invention satisfies this and other needs. The present inventionprovides anti-FcRH5 antibodies that overcome the limitations of currenttherapeutic compositions as well as offer additional advantages thatwill be apparent from the detailed description below.

The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, forthe local delivery of cytotoxic or cytostatic agents, i.e. drugs to killor inhibit tumor cells in the treatment of cancer (Lambert, J. (2005)Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) NatureBiotechnology 23(9):1137-1146; Payne, G. (2003) Cancer Cell 3:207-212;Syrigos and Epenetos (1999) Anticancer Research 19:605-614;Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S.Pat. No. 4,975,278) allows targeted delivery of the drug moiety totumors, and intracellular accumulation therein, where systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15,1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, A. Pinchera et al (ed.s), pp. 475-506). Effortsto improve the therapeutic index, i.e. maximal efficacy and minimaltoxicity of ADC have focused on the selectivity of polyclonal (Rowlandet al (1986) Cancer Immunol. Immunother., 21:183-87) and monoclonalantibodies (mAbs) as well as drug-linking and drug-releasing properties(Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drugmoieties used in antibody drug conjugates include bacterial proteintoxins such as diphtheria toxin, plant protein toxins such as ricin,small molecules such as auristatins, geldanamycin (Mandler et al (2000)J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002)Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al(1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode etal (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine(Rowland et al (1986) supra). The drug moieties may affect cytotoxic andcytostatic mechanisms including tubulin binding, DNA binding, ortopoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

The auristatin peptides, auristatin E (AE) and monomethylauristatin(MMAE), synthetic analogs of dolastatin (WO 02/088172), have beenconjugated as drug moieties to: (i) chimeric monoclonal antibodies cBR96(specific to Lewis Y on carcinomas); (ii) cAC10 which is specific toCD30 on hematological malignancies (Klussman, et al (2004), BioconjugateChemistry 15(4):765-773; Doronina et al (2003) Nature Biotechnology21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465; US2004/0018194; (iii) anti-CD20 antibodies such as rituxan (WO 04/032828)for the treatment of CD20-expressing cancers and immune disorders; (iv)anti-EphB2R antibody 2119 for treatment of colorectal cancer (Mao et al(2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskaret al (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab (HERCEPTIN®, US2005/0238649), and (vi) anti-CD30 antibodies (WO 03/043583). Variants ofauristatin E are disclosed in U.S. Pat. No. 5,767,237 and U.S. Pat. No.6,124,431. Monomethyl auristatin E conjugated to monoclonal antibodiesare disclosed in Senter et al, Proceedings of the American Associationfor Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004. Auristatin analogs MMAE and MMAF have been conjugated to variousantibodies (US 2005/0238649).

Conventional means of attaching, i.e. linking through covalent bonds, adrug moiety to an antibody generally leads to a heterogeneous mixture ofmolecules where the drug moieties are attached at a number of sites onthe antibody. For example, cytotoxic drugs have typically beenconjugated to antibodies through the often-numerous lysine residues ofan antibody, generating a heterogeneous antibody-drug conjugate mixture.Depending on reaction conditions, the heterogeneous mixture typicallycontains a distribution of antibodies with from 0 to about 8, or more,attached drug moieties. In addition, within each subgroup of conjugateswith a particular integer ratio of drug moieties to antibody, is apotentially heterogeneous mixture where the drug moiety is attached atvarious sites on the antibody. Analytical and preparative methods may beinadequate to separate and characterize the antibody-drug conjugatespecies molecules within the heterogeneous mixture resulting from aconjugation reaction. Antibodies are large, complex and structurallydiverse biomolecules, often with many reactive functional groups. Theirreactivities with linker reagents and drug-linker intermediates aredependent on factors such as pH, concentration, salt concentration, andco-solvents. Furthermore, the multistep conjugation process may benonreproducible due to difficulties in controlling the reactionconditions and characterizing reactants and intermediates.

Cysteine thiols are reactive at neutral pH, unlike most amines which areprotonated and less nucleophilic near pH 7. Since free thiol (RSH,sulfhydryl) groups are relatively reactive, proteins with cysteineresidues often exist in their oxidized form as disulfide-linkedoligomers or have internally bridged disulfide groups. Extracellularproteins generally do not have free thiols (Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London,at page 55). Antibody cysteine thiol groups are generally more reactive,i.e. more nucleophilic, towards electrophilic conjugation reagents thanantibody amine or hydroxyl groups. Cysteine residues have beenintroduced into proteins by genetic engineering techniques to formcovalent attachments to ligands or to form new intramolecular disulfidebonds (Better et al (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al(1994) Bioconjugate Chem. 5:126-132; Greenwood et al (1994) TherapeuticImmunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci. USA96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214; Chmuraet al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No.6,248,564). However, engineering in cysteine thiol groups by themutation of various amino acid residues of a protein to cysteine aminoacids is potentially problematic, particularly in the case of unpaired(free Cys) residues or those which are relatively accessible forreaction or oxidation. In concentrated solutions of the protein, whetherin the periplasm of E. coli, culture supernatants, or partially orcompletely purified protein, unpaired Cys residues on the surface of theprotein can pair and oxidize to form intermolecular disulfides, andhence protein dimers or multimers. Disulfide dimer formation renders thenew Cys unreactive for conjugation to a drug, ligand, or other label.Furthermore, if the protein oxidatively forms an intramoleculardisulfide bond between the newly engineered Cys and an existing Cysresidue, both Cys thiol groups are unavailable for active siteparticipation and interactions. Furthermore, the protein may be renderedinactive or non-specific, by misfolding or loss of tertiary structure(Zhang et al (2002) Anal. Biochem. 311:1-9).

Cysteine-engineered antibodies have been designed as FAB antibodyfragments (thioFab) and expressed as full-length, IgG monoclonal(thioMab) antibodies (Junutula, J. R. et al. (2008) J Immunol Methods332:41-52; US 2007/0092940, the contents of which are incorporated byreference). ThioFab and ThioMab antibodies have been conjugated throughlinkers at the newly introduced cysteine thiols with thiol-reactivelinker reagents and drug-linker reagents to prepare antibody drugconjugates (Thio ADC).

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The invention provides anti-FcRH5 antibodies or functional fragmentsthereof, and their method of use in the treatment of hematopoietictumors.

In one aspect, the invention provides an antibody which binds,preferably specifically, to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,antibody fragment, including Fab, Fab′, F(ab′)₂, and Fv fragment,diabody, single domain antibody, chimeric antibody, humanized antibody,single-chain antibody or antibody that competitively inhibits thebinding of an anti-FcRH5 polypeptide antibody to its respectiveantigenic epitope. Antibodies of the present invention may optionally beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, an auristatin, a maytansinoid, adolostatin derivative or a calicheamicin, an antibiotic, a radioactiveisotope, a nucleolytic enzyme, or the like. The antibodies of thepresent invention may optionally be produced in CHO cells or bacterialcells and preferably induce death of a cell to which they bind. Fordetection purposes, the antibodies of the present invention may bedetectably labeled, attached to a solid support, or the like.

In one aspect, the invention provides a humanized anti-FcRH5 antibodywherein the monovalent affinity (e.g. affinity of the antibody as a Fabfragment to FcRH5) or affinity in its bivalent form of the antibody toFcRH5 (e.g. affinity of the antibody as an IgG fragment to FcRH5) issubstantially the same as, lower than, or greater than, the monovalentaffinity or affinity in its bivalent form, respectively, of a murineantibody (e.g. affinity of the murine antibody as a Fab fragment or asan IgG fragment to FcRH5) or a chimeric antibody (e.g. affinity of thechimeric antibody as a Fab fragment or as an IgG fragment to FcRH5),comprising, consisting or consisting essentially of a light chain andheavy chain variable domain sequence as depicted in FIG. 9 (SEQ ID NO:18) and FIG. 10 (SEQ ID NO:20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.4 nM, 0.2nM or 0.5 nM.

In one aspect, an antibody that binds to FcRH5 is provided, wherein theantibody comprises at least one, two, three, four, five or six HVRsselected from the group consisting of:

(i) HVR-L1 comprising the sequence KASQNVGSNVA (SEQ ID NO: 28)

(ii) HVR-L2 comprising the sequence SASYRYS (SEQ ID NO: 29)

(iii) HVR-L3 comprising the sequence QQYKTWT (SEQ ID NO: 30)

(iv) HVR-H1 comprising the sequence GYTFTNYGMN (SEQ ID NO: 37)

(v) HVR-H2 comprising the sequence NTYTGEPTYTDDFKG (SEQ ID NO: 38); and

(vi) HVR-H3 comprising the sequence ARRSIPYYYAMDY (SEQ ID NO: 39). Inone embodiment, the HVR-H1 residue number 9 in a variant HVR-H1 is I. Inone other embodiment, the antibody comprises a HVR-H2 having thesequence of SEQ ID NO: 38. In another embodiment, at least a portion ofthe framework sequence is a human consensus framework sequence. Inanother embodiment, the antibody comprises human κ subgroup 1 consensusframework sequence. In one other embodiment, the antibody comprisesheavy chain human subgroup III consensus framework sequence. In oneother embodiment, the framework sequence comprises a substitution atposition 11, 12, 13, 18, 19, 20, 75, 78, 82, 8=2a, 83, and/or 34. Inanother embodiment, the substitution is Lily, V12K, V13K, L18V, R19K,L20V, K75V, V78A, N82I, N82aS, R83K, and/or M34I. In one otherembodiment, the framework sequence comprises a substitution at position12, 13, 34 and/or 78. In another embodiment, the substitution is V12K,V13K, M34I and/or V78A.

In another embodiment, the antibody comprises a light chain variabledomain having at least 90% amino acid sequence identity to an amino acidsequence selected from SEQ ID NO: 19. In one embodiment, the antibodycomprises a heavy chain variable domain having at least 90% amino acidsequence identity to an amino acid sequence selected from SEQ ID NO: 21.In another embodiment, the antibody comprises a light chain variabledomain having at least 90% amino acid sequence identity to an amino acidsequence selected from SEQ ID NO: 19.

In one other embodiment, the antibody comprises a heavy chain variabledomain having at least 90% amino acid sequence identity to an amino acidsequence selected from SEQ ID NO: 21. In another embodiment, theantibody comprises a heavy chain variable domain having at least 90%amino acid sequence identity to an amino acid sequence selected from SEQID NO: 22. In another embodiment, the antibody comprises a heavy chainvariable domain having at least 90% amino acid sequence identity to anamino acid sequence selected from SEQ ID NO: 23.

In one embodiment, the antibody comprises a heavy chain variable domaincomprising one, two, three or four framework amino acid sequencesselected from SEQ ID NOs: 33, 34, 35 and 36. In other embodiments, theantibody comprises a light chain variable domain comprising one, two,three or four framework amino acid sequences selected from SEQ ID NOs:24, 25, 26, 27, and 28. In another embodiment, the antibody comprises aheavy chain variable domain comprising one, two, three or four frameworkamino acid sequences having at least 90% amino acid sequence identity toan amino acid selected from SEQ ID NOs: 33, 34, 35, and 36. In otherembodiments, the antibody comprises a light chain variable domaincomprising one, two, three or four framework amino acid sequences havingat least 90% amino acid sequence identity to an amino acid selected fromselected from SEQ ID NOs: 24, 25, 26, 27, and 28.

In one aspect, an antibody that binds to FcRH5 is provided, wherein theantibody comprises at least one variant HVR wherein the variant HVRsequence comprises modification of at least one residue of the sequencedepicted in SEQ ID NOs: 28, 29, 30, 37, 38 or 39. In one otherembodiment, the modification is substitution, insertion or deletion.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein monovalent affinity of the antibody to human FcRH5 issubstantially the same as monovalent affinity of a murine antibodycomprising a light chain and heavy chain variable sequence as depictedin FIG. 9 (SEQ ID NO: 18) and FIG. 10 (SEQ ID NO: 20).

In one aspect, the invention provides a humanized anti-FcRH5 antibodywherein monovalent affinity of the antibody to human FcRH5 is at least1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold greater than monovalent affinity ofa murine or chimeric antibody comprising a light chain and heavy chainvariable sequence as depicted in FIG. 9 (SEQ ID NO: 18) and FIG. 10 (SEQID NO: 20).

In one other aspect, the invention provides a humanized anti-FcRH5antibody wherein monovalent affinity of the antibody to human FcRH5 isat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55 or 60-fold lower than monovalentaffinity of a murine or chimeric antibody comprising a light chain andheavy chain variable sequence as depicted in FIG. 9 (SEQ ID NO: 18) andFIG. 10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form tohuman FcRH5 is substantially the same as the affinity of a murineantibody in its bivalent form and comprising a light chain and heavychain variable sequence as depicted in FIG. 9 (SEQ ID NO: 18) and FIG.10 (SEQ ID NO: 20).

In one aspect, the invention provides a humanized anti-FcRH5 antibodythe affinity of the antibody in its bivalent form to human FcRH5 is atleast 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold greater than the affinity ofa murine or chimeric antibody in its bivalent form and comprising alight chain and heavy chain variable sequence as depicted in FIG. 9 (SEQID NO: 18) and FIG. 10 (SEQ ID NO: 20).

In another aspect, the invention provides humanized anti-FcRH5 antibodywherein the affinity of the antibody in its bivalent form to human FcRH5is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60-fold lower than theaffinity of a murine or chimeric antibody in its bivalent formcomprising a light chain and heavy chain variable sequence as depictedin FIG. 9 (SEQ ID NO: 18) and FIG. 10 (SEQ ID NO: 20).

In one other aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form tohuman FcRH5 is 0.4 nM. In one embodiment, the affinity of the antibodyin its bivalent form to human FcRH5 is 0.4 nM+/−0.04.

In one other aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form tohuman FcRH5 is 0.2 nM. In one embodiment, the affinity of the antibodyin its bivalent form to human FcRH5 is 0.2 nM+/−0.02.

In one other aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form tohuman FcRH5 is 0.5 nM. In one embodiment, the affinity of the antibodyin its bivalent form to human FcRH5 is 0.5 nM+/−0.01.

In all aspects, the binding affinity is expressed as a Kd value. Inanother aspect, the binding affinity is measured by Biacore orradioimmunoassay.

In one other aspect, the invention provides a humanized anti-FcRH5antibody wherein the humanized antibody when conjugated to a cytotoxicagent inhibits tumor cell growth.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the humanized antibody when conjugated to a cytotoxicagent inhibits tumor cell growth.

In one embodiment, both the humanized antibody and the chimeric antibodyare monovalent or bivalent. In another embodiment, both the humanizedantibody and chimeric antibody comprise a single Fab region linked to anFc region.

In another aspect, the invention provides an antibody comprising a heavychain variable domain comprising HVR1-HC, HVR2-HC and/or HVR3-HCsequence depicted in FIG. 12 (SEQ ID NOs: 37-39). In one embodiment, thevariable domain comprises FR1-HC, FR2-HC, FR3-HC and/or FR4-HC sequencedepicted in FIG. 12 (SEQ ID NOs: 33-36). In another embodiment, theantibody comprises CH1 and/or Fc sequence depicted in FIG. 12 (SEQ IDNO: 40 and/or 41).

In one other aspect, the invention provides an antibody comprising alight chain variable domain comprising HVR1-LC, HVR2-LC and/or HVR3-LCsequence depicted in FIG. 12 (SEQ ID NOs: 28-30). In one embodiment, thevariable domain comprises FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequencedepicted in FIG. 12 (SEQ ID NOs: 24-27). In another embodiment, theantibody comprises CL1 sequence depicted in FIG. 12 (SEQ ID NO:31).

In one aspect, the invention provides a polypeptide comprising thesequence depicted in FIG. 11 (SEQ ID NO: 21). In another aspect, theinvention provides a polypeptide comprising the sequence depicted inFIG. 9 (SEQ ID NO: 19). In another aspect, the invention provides apolypeptide comprising the sequence depicted in FIG. 11 (SEQ ID NO: 22).In another aspect, the invention provides a polypeptide comprising thesequence depicted in FIG. 11 (SEQ ID NO: 23).

In another aspect, the invention provides an antibody made by theprocess of (a) culturing a cell expressing an antibody comprising aheavy chain variable domain described herein and a light chain variabledomain described herein; and (b) isolating the antibody from saidcultured cell.

In one other aspect, the invention provides an antibody comprising aheavy chain variable domain described herein and a light chain variabledomain described herein. In one embodiment, the antibody is monovalentand comprises an Fc region.

In one aspect, the invention provides a polynucleotide encoding anantibody described herein. In one embodiment, the invention provides avector comprising the polynucleotide. In another embodiment, theinvention provides a host cell comprising the vector.

In one aspect, the invention includes a cysteine engineered anti-FcRH5antibody comprising one or more free cysteine amino acids and a sequenceselected from SEQ ID NOs: 59-62. The cysteine engineered anti-FcRH5antibody may bind to a FcRH5 polypeptide. The cysteine engineeredanti-FcRH5 antibody may be prepared by a process comprising replacingone or more amino acid residues of a parent anti-FcRH5 antibody bycysteine.

In one aspect, the invention includes a cysteine engineered anti-FcRH5antibody comprising one or more free cysteine amino acids wherein thecysteine engineered anti-FcRH5 antibody binds to a FcRH5 polypeptide andis prepared by a process comprising replacing one or more amino acidresidues of a parent anti-FcRH5 antibody by cysteine wherein the parentantibody comprises at least one HVR sequence selected from:

(i) HVR-L1 comprising the sequence KASQNVGSNVA (SEQ ID NO: 28)

(ii) HVR-L2 comprising the sequence SASYRYS (SEQ ID NO: 29)

(iii) HVR-L3 comprising the sequence QQYKTWT (SEQ ID NO: 30)

(iv) HVR-H1 comprising the sequence GYTFTNYGMN (SEQ ID NO: 37)

(v) HVR-H2 comprising the sequence NTYTGEPTYTDDFKG (SEQ ID NO: 38); and

(vi) HVR-H3 comprising the sequence ARRSIPYYYAMDY (SEQ ID NO: 39).

The cysteine engineered anti-FcRH5 antibody may be a monoclonalantibody, antibody fragment, chimeric antibody, humanized antibody,single-chain antibody or antibody that competitively inhibits thebinding of an anti-FcRH5 polypeptide antibody to its respectiveantigenic epitope. Antibodies of the present invention may optionally beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, an auristatin or maytansinoid. Theantibodies of the present invention may optionally be produced in CHOcells or bacterial cells and preferably inhibit the growth orproliferation of or induce the death of a cell to which they bind. Fordiagnostic purposes, the antibodies of the present invention may bedetectably labeled, attached to a solid support, or the like.

In one aspect, the invention provides methods for making an antibody ofthe invention. For example, the invention provides a method of making aFcRH5 antibody (which, as defined herein includes full length andfragments thereof), said method comprising expressing in a suitable hostcell a recombinant vector of the invention encoding said antibody (orfragment thereof), and recovering said antibody.

In another aspect, the invention provides a bispecific antibody capableof binding to a first cell that expresses FcRH5 and to a second cellthat expresses a cell-surface target antigen. In one embodiment, thesecond cell is a T cell. In one embodiment, the cell-surface targetantigen is CD3. In certain embodiments, the bispecific antibody is aprotruberance-into-cavity antibody. In one embodiment, the bispecificantibody is aglycosylated. In one embodiment, the bispecific antibody isproduced in an Escherichia coli host cell. In one embodiment, thebispecific antibody lacks one or more Fc effector functions. In oneembodiment, the bispecific antibody lacks ADCC activity.

In one aspect, the invention is a pharmaceutical formulation comprisingan antibody of the invention or an antibody-drug conjugate of theinvention, and a pharmaceutically acceptable diluent, carrier orexcipient.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more FcRH5antibodies of the invention.

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more FcRH5 antibodies of theinvention; and a second container comprising a buffer.

In one aspect, the invention provides use of a FcRH5 antibody of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor and/or acell proliferative disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumorand/or a cell proliferative disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor and/or a cellproliferative disorder.

In one aspect, the invention provides a method of inhibiting the growthof a cell that expresses FcRH5, said method comprising contacting saidcell with an antibody of the invention thereby causing an inhibition ofgrowth of said cell. In one embodiment, the antibody is conjugated to acytotoxic agent. In one embodiment, the antibody is conjugated to agrowth inhibitory agent.

In one aspect, the invention provides a method of therapeuticallytreating a mammal having a cancerous tumor comprising a cell thatexpresses FcRH5, said method comprising administering to said mammal atherapeutically effective amount of an antibody of the invention,thereby effectively treating said mammal. In one embodiment, theantibody is conjugated to a cytotoxic agent. In one embodiment, theantibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression of FcRH5, said method comprising administering to a subjectin need of such treatment an effective amount of an antibody of theinvention, thereby effectively treating or preventing said cellproliferative disorder. In one embodiment, said proliferative disorderis cancer. In one embodiment, the antibody is conjugated to a cytotoxicagent. In one embodiment, the antibody is conjugated to a growthinhibitory agent.

In one aspect, the invention provides a method for inhibiting the growthof a cell, wherein growth of said cell is at least in part dependentupon a growth potentiating effect of FcRH5, said method comprisingcontacting said cell with an effective amount of an antibody of theinvention, thereby inhibiting the growth of said cell. In oneembodiment, the antibody is conjugated to a cytotoxic agent. In oneembodiment, the antibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method of therapeuticallytreating a tumor in a mammal, wherein the growth of said tumor is atleast in part dependent upon a growth potentiating effect of FcRH5, saidmethod comprising contacting said cell with an effective amount of anantibody of the invention, thereby effectively treating said tumor. Inone embodiment, the antibody is conjugated to a cytotoxic agent. In oneembodiment, the antibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method of treating cancercomprising administering to a patient the pharmaceutical formulationcomprising an immunoconjugate described herein, acceptable diluent,carrier or excipient.

In one aspect, the invention provides a method of inhibiting B cellproliferation comprising exposing a cell to an immunoconjugatecomprising an antibody of the invention under conditions permissive forbinding of the immunoconjugate to FcRH5.

The methods of the present invention can further comprise additionaltreatment steps. For example, in one embodiment, a method furthercomprises a step wherein a targeted cell and/or tissue (e.g., a cancercell) is exposed to radiation treatment or a chemotherapeutic agent.

In one aspect, the invention provides methods comprising administrationof an effective amount of an anti-FcRH5 antibody in combination with aneffective amount of another therapeutic agent (such as ananti-angiogenesis agent, another antibody, a chemotherapeutic agent, acytotoxic agent, an immunosuppressive agent, a prodrug, a cytokine,cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancervaccine, an analgesic, or a growth inhibitory agent). For example,anti-FcRH5 antibodies or immunoconjugates are used in combinations withan anti-cancer agent or an anti-angiogenic agent to treat variousneoplastic or non-neoplastic conditions. In particular examples, theanti-FcRH5 antibodies are used in combination with Velcade®(bortezomib), Revlimid® (lenalidomide), tamoxifen, letrozole,exemestane, anastrozole, irinotecan, cetuximab, fulvestrant,vinorelbine, bevacizumab, vincristine, cisplatin, gemcitabine,methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel,pemetrexed, 5-fluorouracil, doxorubicin, bortezomib, lenalidomide,dexamethasone, melphalin, prednisone, vincristine, thalidomide.

Depending on the specific cancer indication to be treated, thecombination therapy of the invention can be combined with additionaltherapeutic agents, such as chemotherapeutic agents, or additionaltherapies such as radiotherapy or surgery. Many known chemotherapeuticagents can be used in the combination therapy of the invention.Preferably those chemotherapeutic agents that are standard for thetreatment of the specific indications will be used. Dosage or frequencyof each therapeutic agent to be used in the combination is preferablythe same as, or less than, the dosage or frequency of the correspondingagent when used without the other agent(s).

In another aspect, the invention provides any of the anti-FcRH5antibodies described herein, wherein the anti-FcRH5 antibody comprises adetectable label.

In one aspect, the invention provides a method of determining thepresence of FcRH5 in a sample suspected of containing FcRH5, said methodcomprising exposing said sample to an antibody of the invention, anddetermining binding of said antibody to FcRH5 in said sample whereinbinding of said antibody to FcRH5 in said sample is indicative of thepresence of said protein in said sample.

In one aspect, the invention provides a method of diagnosing a cellproliferative disorder associated with an increase in cells, such as Bcells, expressing FcRH5 is provided, the method comprising contacting atest cells in a biological sample with any of the above antibodies;determining the level of antibody bound to test cells in the sample bydetecting binding of the antibody to FcRH5; and comparing the level ofantibody bound to cells in a control sample, wherein the level ofantibody bound is normalized to the number of FcRH5-expressing cells inthe test and control samples, and wherein a higher level of antibodybound in the test sample as compared to the control sample indicates thepresence of a cell proliferative disorder associated with cellsexpressing FcRH5.

In one aspect, the invention provides a method of detecting solubleFcRH5 in blood or serum, the method comprising contacting a test sampleof blood or serum from a mammal suspected of experiencing a B cellproliferative disorder with an anti-FcRH5 antibody of the invention anddetecting a increase in soluble FcRH5 in the test sample relative to acontrol sample of blood or serum from a normal mammal.

In one aspect, the invention provides a method of binding an antibody ofthe invention to a cell that expresses FcRH5, said method comprisingcontacting said cell with an antibody of the invention. In oneembodiment, the antibody is conjugated to a cytotoxic agent. In oneembodiment, the antibody is conjugated to a growth inhibitory agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts chimeric anti-human FcRH5 7D11 light chain (DNA).

FIG. 2 depicts chimeric anti-human FcRH5 7D11 light chain (amino acid).

FIG. 3 depicts chimeric anti-human FcRH5 7D11 heavy chain (DNA).

FIG. 4 depicts chimeric anti-human FcRH5 7D11 heavy chain (amino acid).

FIG. 5 depicts chimeric anti-human FcRH5 (mAb13G9) light chain (DNA)(SEQ ID NO: 52).

FIG. 6 depicts chimeric anti-human FcRH5 (mAb13G9) light chain (aminoacid) (SEQ ID NO: 53).

FIG. 7 depicts chimeric anti-human FcRH5 (mAb13G9) heavy chain (DNA)(SEQ ID NO: 54).

FIG. 8 depicts chimeric anti-human FcRH5 (mAb13G9) heavy chain (aminoacid) (SEQ ID NO: 55).

FIG. 9 shows the alignment of variable light chains for murine 13G9 (SEQID NO: 18) and humanized 13G9v1 (SEQ ID NO: 19). FIG. 9 discloses the“hum k1” sequence as SEQ ID NO: 56.

FIG. 10 shows the alignment of variable heavy chains for murine 13G9(SEQ ID NO: 20) and humanized 13G9v1 (SEQ ID NO: 57). FIG. 10 disclosesthe “hum III” sequence as SEQ ID NO: 58.

FIG. 11 depicts the alignment of variable heavy chains for humanized13G9v1 (SEQ ID NO: 21), humanized 13G9v3 (SEQ ID NO: 22) and humanized13G9v8 (SEQ ID NO: 23).

FIG. 12 shows amino acid sequences of an antibody of the invention(hu13G9 v1). FIG. 12 discloses SEQ ID NOS 24-42, respectively, in orderof appearance.

FIG. 13 shows amino acid sequences of cysteine engineered polypeptidechains of a thio-Mab of the invention. FIG. 13 discloses SEQ ID NOS 59and 60, respectively, in order of appearance.

FIG. 14 shows amino acid sequences of cysteine engineered polypeptidechains of a thio-Mab of the invention. FIG. 14 discloses SEQ ID NOS 61and 62, respectively, in order of appearance.

FIG. 15 shows crossreactivity of FcRH5 antibodies.

FIG. 16 shows crossreactivity of FcRH5 antibodies.

FIG. 17 shows crossreactivity of FcRH5 antibodies.

FIG. 18 shows FcRH5 antibody titration.

FIG. 19 shows FcRH5 antibody titration.

FIG. 20 shows the effect of antibodies of the invention on mean tumorvolume.

FIG. 21 shows the effect of antibodies of the invention on mean bodyweight.

FIG. 22 shows the effect of antibodies of the invention on mean tumorvolume.

FIG. 23 shows the effect of antibodies of the invention on mean bodyweight.

FIG. 24 shows a single compound dose response analysis.

FIG. 25 shows a single compound dose response analysis.

FIG. 26 shows a nucleotide sequence (SEQ ID NO: 63) of a PRO820 cDNA,wherein SEQ ID NO: 63 is a clone designated herein as “DNA56041-1416”(also referred here in as “FcRH5”). The nucleotide sequence encodes forFcRH5 with the start and stop codons shown in bold and underlined. (SeeGoddard et al. U.S. Pat. No. 7,491,529).

FIG. 27 shows the amino acid sequence (SEQ ID NO: 64) derived from thecoding sequence of SEQ ID NO: 63 shown in FIG. 26. (See Goddard et al.U.S. Pat. No. 7,491,529).

FIG. 28A-B shows a nucleotide sequence (SEQ ID NO: 65) of a PRO52387cDNA, wherein SEQ ID NO: 65 is a clone designated herein as “DNA257845”(also referred here in as “FcRH5”). The nucleotide sequence encodes forFcRH5 with the start and stop codons shown in bold and underlined (SeeChang et al. U.S. Published Patent Application No. 20060251662).

FIG. 29 shows the amino acid sequence (SEQ ID NO: 66) derived from thecoding sequence of SEQ ID NO: 65 shown in FIG. 28A-B (See Chang et al.U.S. Published Patent Application No. 20060251662).

FIG. 30 shows the nucleotide sequence (SEQ ID NO:67) of a PRO314992cDNA, wherein SEQ ID NO: 67 is a clone designated herein as “DNA676969”from cynomologous monkey (also referred to herein as “cyno FcRH5”).

FIG. 31 shows the amino acid sequence (SEQ ID NO: 68) derived from thecoding sequence of SEQ ID NO: 67 shown in FIG. 30.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention provides methods, compositions, kits and articles ofmanufacture for identifying compositions useful for the treatment ofhematopoietic tumor in mammals and to methods of using thosecompositions of matter for the same.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

I. GENERAL TECHNIQUES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001).

II. DEFINITIONS

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth conflicts with any document incorporated hereinby reference, the definition set forth below shall control.

A “B-cell surface marker” or “B-cell surface antigen” herein is anantigen expressed on the surface of a B cell that can be targeted withan antagonist that binds thereto, including but not limited to,antibodies to a B-cell surface antigen or a soluble form a B-cellsurface antigen capable of antagonizing binding of a ligand to thenaturally occurring B-cell antigen. Exemplary B-cell surface markersinclude the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53,CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81,CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers (fordescriptions, see The Leukocyte Antigen Facts Book, 2^(nd) Edition.1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., NewYork). Other B-cell surface markers include RP105, FcRH2, B-cell CR2,CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14,SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287.The B-cell surface marker of particular interest is preferentiallyexpressed on B cells compared to other non-B-cell tissues of a mammaland may be expressed on both precursor B cells and mature B cells.

The term “FcRH5”, as used herein, refers to any native FcRH5 from anyvertebrate source, including mammals such as primates (e.g. humans,cynomologus monkey (cyno)) and rodents (.e.g., mice and rats), unlessotherwise indicated. Human FcRH5 is also referred herein to as “TAHO18”or “PRO85143” (SEQ ID NO: 2) and encoded by the nucleotide sequence (SEQID NO: 1) also referred herein to as “DNA340394”. Cynomologus FcRH5 isalso referred herein to as “cyno FcRH5”. The term “FcRH5” encompasses“full-length,” unprocessed FcRH5 as well as any form of FcRH5 thatresults from processing in the cell. The term also encompasses naturallyoccurring variants of FcRH5, e.g., splice variants, allelic variants andisoforms. The FcRH5 polypeptides described herein may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant or synthetic methods. A “nativesequence FcRH5 polypeptide” comprises a polypeptide having the sameamino acid sequence as the corresponding FcRH5 polypeptide derived fromnature. Such native sequence FcRH5 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence FcRH5 polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific FcRH5polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In certainembodiments of the invention, the native sequence FcRH5 polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons (if indicated) are shown inbold font and underlined in the figures. Nucleic acid residues indicatedas “N” in the accompanying figures are any nucleic acid residue.However, while the FcRH5 polypeptides disclosed in the accompanyingfigures are shown to begin with methionine residues designated herein asamino acid position 1 in the figures, it is conceivable and possiblethat other methionine residues located either upstream or downstreamfrom the amino acid position 1 in the figures may be employed as thestarting amino acid residue for the FcRH5 polypeptides.

“mu7D11” or “MA7D11” or “murine FcRH5 (7D11) antibody” or “murineanti-FcRH5 (7D11) antibody” is used herein to specifically refer tomurine anti-FcRH5 monoclonal antibody wherein the murine anti-FcRH5monoclonal antibody comprises the light chain variable domain of FIG. 9(SEQ ID NO: 18) and FIG. 10 (SEQ ID NO: 20). Murine anti-FcRH5monoclonal antibody may be purchased from commercial sources.

“ch13G9” or “chMAFcRH5 (13G9)” or “chimeric MAFcRH5 (13G9) antibody” isused herein to specifically refer to chimeric anti-human FcRH5 (13G)antibody wherein the chimeric anti-FcRH5 antibody comprises the lightchain of SEQ ID NO:53 (FIG. 6), which comprises the light chain constantdomain of human IgG1. The chimeric anti-FcRH5 (13G9) antibody furthercomprises the heavy chain of SEQ ID NO:55 (FIG. 8), which comprises theconstant domain of human IgG1.

“anti-cynoFcRH5” or “anti-cyno FcRH5” is used herein to refer toantibodies that binds to cyno FcRH5.

“13G9-graft” or “13G9-grafted ‘humanized’ antibody” or “hu13G9 graft” isused herein to specifically refer to the graft generated by grafting thehypervariable regions from murine 13G9 anti-FcRH5 antibody (MA7D11) intothe acceptor human consensus VL kappa I (huKI) and human subgroup IIIconsensus VH (huIII) with R71A, N73T and L78A (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992)) (See Example section 1 and FIGS.9-12). “hu13G9” or “hu13G9 v1” or “hu13G9 v3” or “hu13G9 v8” is usedherein to specifically refer to humanized 13G9 antibody.

A “modification” of an amino acid residue/position, as used herein,refers to a change of a primary amino acid sequence as compared to astarting amino acid sequence, wherein the change results from a sequencealteration involving said amino acid residue/positions. For example,typical modifications include substitution of the residue (or at saidposition) with another amino acid (e.g., a conservative ornon-conservative substitution), insertion of one or more (generallyfewer than 5 or 3) amino acids adjacent to said residue/position, anddeletion of said residue/position. An “amino acid substitution”, orvariation thereof, refers to the replacement of an existing amino acidresidue in a predetermined (starting) amino acid sequence with adifferent amino acid residue. Generally and preferably, the modificationresults in alteration in at least one physicobiochemical activity of thevariant polypeptide compared to a polypeptide comprising the starting(or “wild type”) amino acid sequence. For example, in the case of anantibody, a physicobiochemical activity that is altered can be bindingaffinity, binding capability and/or binding effect upon a targetmolecule.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-FcRH5 monoclonal antibodies (includingagonist, antagonist, neutralizing antibodies, full length or intactmonoclonal antibodies), anti-FcRH5 antibody compositions withpolyepitopic specificity, polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies so long as theyexhibit the desired biological activity), formed from at least twointact antibodies, single chain anti-FcRH5 antibodies, and fragments ofanti-FcRH5 antibodies (see below), including Fab, Fab′, F(ab′)₂ and Fvfragments, diabodies, single domain antibodies (sdAbs), as long as theyexhibit the desired biological or immunological activity. The term“immunoglobulin” (Ig) is used interchangeable with antibody herein. Anantibody can be human, humanized and/or affinity matured.

The term “anti-FcRH5 antibody” or “an antibody that binds to FcRH5”refers to an antibody that is capable of binding FcRH5 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting FcRH5. Preferably, the extent of bindingof an anti-FcRH5 antibody to an unrelated, non-FcRH5 protein is lessthan about 10% of the binding of the antibody to FcRH5 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to FcRH5 has a dissociation constant (Kd) of ≦1 μM, ≦100 nM,≦10 nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, anti-FcRH5 antibodybinds to an epitope of FcRH5 that is conserved among FcRH5 fromdifferent species.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for a and c isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting a3-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H)1,C_(H)2 and C_(H)3. The constant domains may be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. Preferably, the intact antibody has one ormore effector functions.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments. In one embodiment, an antibody fragmentcomprises an antigen binding site of the intact antibody and thusretains the ability to bind antigen.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H)1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. From the folding of these two domains emanate six hypervariableloops (3 loops each from the H and L chain) that contribute the aminoacid residues for antigen binding and confer antigen binding specificityto the antibody. However, even a single variable domain (or half of anFv comprising only three CDRs specific for an antigen) has the abilityto recognize and bind antigen, although at a lower affinity than theentire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). The small antibody fragments are prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Diabodies may be bivalent or bispecific.Bispecific diabodies are heterodimers of two “crossover” sFv fragmentsin which the V_(H) and V_(L) domains of the two antibodies are presenton different polypeptide chains. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; Hudson et al., Nat. Med. 9:129-134(2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993). Triabodies and tetrabodies are also described in Hudson et al.,Nat. Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. Soc.Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.,5:428-433 (1994).

“Thio” when used herein to refer to an antibody refers to acysteine-engineered antibody while “hu” when used herein to refer to anantibody refers to a humanized antibody.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the V11 (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop whennumbered using the Kabat numbering convention varies between 1132 and1134 depending on the length of the loop (this is because the Kabatnumbering scheme places the insertions at H35A and H₃₅B; if neither 35Anor 35B is present, the loop ends at 32; if only 35A is present, theloop ends at 33; if both 35A and 35B are present, the loop ends at 34).The AbM hypervariable regions represent a compromise between the KabatCDRs and Chothia structural loops, and are used by Oxford Molecular'sAbM antibody modeling software. The “contact” hypervariable regions arebased on an analysis of the available complex crystal structures. Theresidues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L24-L34 L30-L36 L2L50-L56 L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 H1H31-H35B H26-H35B H26-H32 . . . 34 H30-H35B (Kabat Numbering) H1 H31-H35H26-H35 H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35B (H1), 50-65, 47-65 or 49-65 (112) and 93-102, 94-102 or95-102 (H3) in the VH. The variable domain residues are numberedaccording to Kabat et al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody. Unless stated otherwiseherein, references to residue numbers in the variable domain ofantibodies means residue numbering by the Kabat numbering system. Unlessstated otherwise herein, references to residue numbers in the constantdomain of antibodies means residue numbering by the EU numbering system(e.g., see U.S. Provisional Application No. 60/640,323, Figures for EUnumbering).

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of HVRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “species-dependent antibody,” e.g., a mammalian anti-human IgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

“Or better” when used herein to refer to binding affinity refers to astronger binding between a molecule and its binding partner. “Or better”when used herein refers to a stronger binding, represented by a smallernumerical Kd value. For example, an antibody which has an affinity foran antigen of “0.6 nM or better”, the antibody's affinity for theantigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any valueless than 0.6 nM.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays.

According to another embodiment the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) is calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. MolBiol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surfaceplasmon resonance assay above, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) as described above.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of statistical significance within the context ofthe biological characteristic measured by said values (e.g., Kd values,HAMA response). The difference between said two values is preferablygreater than about 10%, preferably greater than about 20%, preferablygreater than about 30%, preferably greater than about 40%, preferablygreater than about 50% as a function of the value for thereference/comparator antibody.

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework, or from a human consensus framework.An acceptor human framework “derived from” a human immunoglobulinframework or human consensus framework may comprise the same amino acidsequence thereof, or may contain pre-existing amino acid sequencechanges. Where pre-existing amino acid changes are present, preferablyno more than 5 and preferably 4 or less, or 3 or less, pre-existingamino acid changes are present. Where pre-existing amino acid changesare present in a VH, preferably those changes are only at three, two orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:14)-H1-WVRQAPGKGLEWV (SEQ ID NO: 15)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO: 16)-H3-WGQGTLVTVSS (SEQ ID NO: 17).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VL subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 43)- DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 44)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 45)- L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 46)) L3-FGQGTKVEIKR. 

An “unmodified human framework” is a human framework which has the sameamino acid sequence as the acceptor human framework, e.g. lacking humanto non-human amino acid substitution(s) in the acceptor human framework.

An “altered hypervariable region” for the purposes herein is ahypervariable region comprising one or more (e.g. one to about 16) aminoacid substitution(s) therein.

An “un-modified hypervariable region” for the purposes herein is ahypervariable region having the same amino acid sequence as a non-humanantibody from which it was derived, i.e. one which lacks one or moreamino acid substitutions therein.

An antibody “which binds” an antigen of interest, e.g. atumor-associated polypeptide antigen target, is one that binds theantigen with sufficient affinity such that the antibody is useful as atherapeutic agent in targeting a cell or tissue expressing the antigen,and does not significantly cross-react with other proteins. In suchembodiments, the extent of binding of the antibody to a “non-target”protein will be less than about 10% of the binding of the antibody toits particular target protein as determined by fluorescence activatedcell sorting (FACS) analysis or radioimmunoprecipitation (RIA). Withregard to the binding of an antibody to a target molecule, the term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetmeans binding that is measurably different from a non-specificinteraction. Specific binding can be measured, for example, bydetermining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. For example, specific binding can bedetermined by competition with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. The term“specific binding” or “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide targetas used herein can be exhibited, for example, by a molecule having a Kdfor the target of at least about 10⁻⁴ M, alternatively at least about10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternatively at leastabout 10⁻⁷ M, alternatively at least about 10⁻⁸ M, alternatively atleast about 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, alternativelyat least about 10⁻¹¹ M, alternatively at least about 10⁻¹² M, orgreater. In one embodiment, the term “specific binding” refers tobinding where a molecule binds to a particular polypeptide or epitope ona particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

An antibody that “inhibits the growth of tumor cells expressing a FcRH5polypeptide” or a “growth inhibitory” antibody is one which results inmeasurable growth inhibition of cancer cells expressing oroverexpressing the appropriate FcRH5 polypeptide. The FcRH5 polypeptidemay be a transmembrane polypeptide expressed on the surface of a cancercell or may be a polypeptide that is produced and secreted by a cancercell. Preferred growth inhibitory anti-FcRH5 antibodies inhibit growthof FcRH5-expressing tumor cells by greater than 20%, preferably fromabout 20% to about 50%, and even more preferably, by greater than 50%(e.g., from about 50% to about 100%) as compared to the appropriatecontrol, the control typically being tumor cells not treated with theantibody being tested. In one embodiment, growth inhibition can bemeasured at an antibody concentration of about 0.1 to 30 μg/ml or about0.5 nM to 200 nM in cell culture, where the growth inhibition isdetermined 1-10 days after exposure of the tumor cells to the antibody.Growth inhibition of tumor cells in vivo can be determined in variousways such as is described in the Experimental Examples section below.The antibody is growth inhibitory in vivo if administration of theanti-FcRH5 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses a FcRH5 polypeptide. Preferablythe cell is a tumor cell, e.g., a hematopoietic cell, such as a B cell,T cell, basophil, eosinophil, neutrophil, monocyte, platelet orerythrocyte. Various methods are available for evaluating the cellularevents associated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells. Preferably, the antibody which induces apoptosis isone which results in about 2 to 50 fold, preferably about 5 to 50 fold,and most preferably about 10 to 50 fold, induction of annexin bindingrelative to untreated cell in an annexin binding assay.

An antibody which “induces cell death” is one which causes a viable cellto become nonviable. The cell is one which expresses a FcRH5 polypeptideand is of a cell type which specifically expresses or overexpresses aFcRH5 polypeptide. The cell may be cancerous or normal cells of theparticular cell type. The FcRH5 polypeptide may be a transmembranepolypeptide expressed on the surface of a cancer cell or may be apolypeptide that is produced and secreted by a cancer cell. The cell maybe a cancer cell, e.g., a B cell or T cell. Cell death in vitro may bedetermined in the absence of complement and immune effector cells todistinguish cell death induced by antibody-dependent cell-mediatedcytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus,the assay for cell death may be performed using heat inactivated serum(i.e., in the absence of complement) and in the absence of immuneeffector cells. To determine whether the antibody is able to induce celldeath, loss of membrane integrity as evaluated by uptake of propidiumiodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11(1995)) or 7AAD can be assessed relative to untreated cells. Preferredcell death-inducing antibodies are those which induce PI uptake in thePI uptake assay in BT474 cells.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include C lq binding;CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays as disclosed, for example, in definitions herein.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification, preferably one or more amino acid substitution(s).Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% homology with a native sequence Fc region and/or withan Fc region of a parent polypeptide, and most preferably at least about90% homology therewith, more preferably at least about 95% homologytherewith.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FeγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. See also, e.g., Shields et al. J. Biol.Chem. 9(2):6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Polypeptidevariants with altered Fc region amino acid sequences (polypeptides witha variant Fc region) and increased or decreased C lq binding capabilityare described, e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642.See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising antibody” refers to an antibody thatcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the antibody or by recombinant engineering of thenucleic acid encoding the antibody. Accordingly, a compositioncomprising an antibody having an Fc region according to this inventioncan comprise an antibody with K447, with all K447 removed, or a mixtureof antibodies with and without the K447 residue.

The FcRH5 polypeptide “extracellular domain” or “ECD” refers to a formof the FcRH5 polypeptide which is essentially free of the transmembraneand cytoplasmic domains. Ordinarily, a FcRH5 polypeptide ECD will haveless than 1% of such transmembrane and/or cytoplasmic domains andpreferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the FcRH5polypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a FcRH5 polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are contemplated by thepresent invention.

The approximate location of the “signal peptides” of the FcRH5polypeptide disclosed herein may be shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“FcRH5 polypeptide variant” means a FcRH5 polypeptide, preferably anactive FcRH5 polypeptide, as defined herein having at least about 80%amino acid sequence identity with a full-length native sequence FcRH5polypeptide sequence as disclosed herein, a FcRH5 polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a FcRH5 polypeptide, with or without the signal peptide, as disclosedherein or any other fragment of a full-length FcRH5 polypeptide sequenceas disclosed herein (such as those encoded by a nucleic acid thatrepresents only a portion of the complete coding sequence for afull-length FcRH5 polypeptide). Such FcRH5 polypeptide variants include,for instance, FcRH5 polypeptides wherein one or more amino acid residuesare added, or deleted, at the N- or C-terminus of the full-length nativeamino acid sequence. Ordinarily, a FcRH5 polypeptide variant will haveat least about 80% amino acid sequence identity, alternatively at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to afull-length native sequence FcRH5 polypeptide sequence as disclosedherein, a FcRH5 polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a FcRH5 polypeptide, withor without the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length FcRH5 polypeptidesequence as disclosed herein. Ordinarily, FcRH5 variant polypeptides areat least about 10 amino acids in length, alternatively at least about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600 amino acids in length, or more. Optionally, FcRH5 variantpolypeptides will have no more than one conservative amino acidsubstitution as compared to the native FcRH5 polypeptide sequence,alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservativeamino acid substitution as compared to the native FcRH5 polypeptidesequence.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence, i.e. FcRH5 polypeptide sequences identifiedherein, is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific peptide or polypeptide sequence, i.e. FcRH5 polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Forpurposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 1 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 1 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

“FcRH5 variant polynucleotide” or “FcRH5 variant nucleic acid sequence”means a nucleic acid molecule which encodes a FcRH5 polypeptide,preferably an active FcRH5 polypeptide, as defined herein and which hasat least about 80% nucleic acid sequence identity with a nucleotide acidsequence encoding a full-length native sequence FcRH5 polypeptidesequence as disclosed herein, a full-length native sequence FcRH5polypeptide sequence lacking the signal peptide as disclosed herein, anextracellular domain of a FcRH5 polypeptide, with or without the signalpeptide, as disclosed herein or any other fragment of a full-lengthFcRH5 polypeptide sequence as disclosed herein (such as those encoded bya nucleic acid that represents only a portion of the complete codingsequence for a full-length FcRH5 polypeptide). Ordinarily, a FcRH5variant polynucleotide will have at least about 80% nucleic acidsequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%nucleic acid sequence identity with a nucleic acid sequence encoding afull-length native sequence FcRH5 polypeptide sequence as disclosedherein, a full-length native sequence FcRH5 polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of aFcRH5 polypeptide, with or without the signal sequence, as disclosedherein or any other fragment of a full-length FcRH5 polypeptide sequenceas disclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, FcRH5 variant polynucleotides are at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000,1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120,1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240,1250, 1260, 1270, 1280, 1290, or 1300 nucleotides in length, wherein inthis context the term “about” means the referenced nucleotide sequencelength plus or minus 10% of that referenced length.

“Percent (%) nucleic acid sequence identity” with respect toFcRH5-encoding nucleic acid sequences identified herein is defined asthe percentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the FcRH5 nucleic acid sequence of interest,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. Unless specifically stated otherwise, all % nucleicacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In other embodiments, FcRH5 variant polynucleotides are nucleic acidmolecules that encode a FcRH5 polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length FcRH5polypeptide as disclosed herein. FcRH5 variant polypeptides may be thosethat are encoded by a FcRH5 variant polynucleotide.

The term “full-length coding region” when used in reference to a nucleicacid encoding a FcRH5 polypeptide refers to the sequence of nucleotideswhich encode the full-length FcRH5 polypeptide of the invention (whichis often shown between start and stop codons, inclusive thereof, in theaccompanying figures). The term “full-length coding region” when used inreference to an ATCC deposited nucleic acid refers to the FcRH5polypeptide-encoding portion of the cDNA that is inserted into thevector deposited with the ATCC (which is often shown between start andstop codons, inclusive thereof, in the accompanying figures (start andstop codons are bolded and underlined in the figures).

“Isolated,” when used to describe the various FcRH5 polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the FcRH5 polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” FcRH5 polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50EC;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42EC; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42EC, with a 10minute wash at 42EC in 0.2×SSC (sodium chloride/sodium citrate) followedby a 10 minute high-stringency wash consisting of 0.1×SSC containingEDTA at 55EC.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37EC in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50EC. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a FcRH5 polypeptide or anti-FcRH5 antibody fusedto a “tag polypeptide”. The tag polypeptide has enough residues toprovide an epitope against which an antibody can be made, yet is shortenough such that it does not interfere with activity of the polypeptideto which it is fused. The tag polypeptide preferably also is fairlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 8 and 50 amino acidresidues (preferably, between about 10 and 20 amino acid residues).

“Active” or “activity” for the purposes herein refers to form(s) of aFcRH5 polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring FcRH5, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring FcRH5 other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring FcRH5 and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring FcRH5.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native FcRH5 polypeptide. In a similar manner,the term “agonist” is used in the broadest sense and includes anymolecule that mimics a biological activity of a native FcRH5polypeptide. Suitable agonist or antagonist molecules specificallyinclude agonist or antagonist antibodies or antibody fragments,fragments or amino acid sequence variants of native FcRH5 polypeptides,peptides, antisense oligonucleotides, small organic molecules, etc.Methods for identifying agonists or antagonists of a FcRH5 polypeptide,may comprise contacting a FcRH5 polypeptide, with a candidate agonist orantagonist molecule and measuring a detectable change in one or morebiological activities normally associated with the FcRH5 polypeptide.

“Purified” means that a molecule is present in a sample at aconcentration of at least 95% by weight, or at least 98% by weight ofthe sample in which it is contained.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromasomally or at achromosomal location that is different from its natural chromosomallocation.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, .alpha.-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR.sub.2 (“amidate”), P(O)R,P(O)OR′, CO or CH.sub.2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C.)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, hematopoietic cancers or blood-related cancers, such as lymphoma,leukemia, myeloma or lymphoid malignancies, but also cancers of thespleen and cancers of the lymph nodes and also carcinoma, blastoma andsarcoma. More particular examples of cancer include B-cell associatedcancers, including for example, high, intermediate and low gradelymphomas (including B cell lymphomas such as, for example,mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin'slymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, smalllymphocytic lymphoma, marginal zone lymphoma, diffuse large celllymphoma, follicular lymphoma, and Hodgkin's lymphoma and T celllymphomas) and leukemias (including secondary leukemia, chroniclymphocytic leukemia (CLL), such as B cell leukemia (CD5± Blymphocytes), myeloid leukemia, such as acute myeloid leukemia, chronicmyeloid leukemia, lymphoid leukemia, such as acute lymphoblasticleukemia (ALL) and myelodysplasia), and other hematological and/or Bcell- or T-cell-associated cancers. Also included are cancers ofadditional hematopoietic cells, including polymorphonuclear leukocytes,such as basophils, eosinophils, neutrophils and monocytes, dendriticcells, platelets, erythrocytes and natural killer cells. Also includedare cancerous B cell proliferative disorders selected from thefollowing: lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma. The origins ofB-cell cancers include as follows: marginal zone B-cell lymphoma originsin memory B-cells in marginal zone, follicular lymphoma and diffuselarge B-cell lymphoma originates in centrocytes in the light zone ofgerminal centers, chronic lymphocytic leukemia and small lymphocyticleukemia originates in B1 cells (CD5±), mantle cell lymphoma originatesin naive B-cells in the mantle zone and Burkitt's lymphoma originates incentroblasts in the dark zone of germinal centers. Tissues which includehematopoietic cells referred herein to as “hematopoietic cell tissues”include thymus and bone marrow and peripheral lymphoid tissues, such asspleen, lymph nodes, lymphoid tissues associated with mucosa, such asthe gut-associated lymphoid tissues, tonsils, Peyer's patches andappendix and lymphoid tissues associated with other mucosa, for example,the bronchial linings. Further particular examples of such cancersinclude squamous cell cancer, small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastrointestinalcancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer,liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

A “B-cell malignancy” herein includes non-Hodgkin's lymphoma (NHL),including low grade/follicular NHL, small lymphocytic (SL) NHL,intermediate grade/follicular NHL, intermediate grade diffuse NHL, highgrade immunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma,AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia,non-Hodgkin's lymphoma (NHL), lymphocyte predominant Hodgkin's disease(LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia(CLL), indolent NHL including relapsed indolent NHL andrituximab-refractory indolent NHL; leukemia, including acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairycell leukemia, chronic myeloblastic leukemia; mantle cell lymphoma; andother hematologic malignancies. Such malignancies may be treated withantibodies directed against B-cell surface markers, such as FcRH5. Suchdiseases are contemplated herein to be treated by the administration ofan antibody directed against a B cell surface marker, such as FcRH5, andincludes the administration of an unconjugated (“naked”) antibody or anantibody conjugated to a cytotoxic agent as disclosed herein. Suchdiseases are also contemplated herein to be treated by combinationtherapy including an anti-FcRH5 antibody or anti-FcRH5 antibody drugconjugate of the invention in combination with another antibody orantibody drug conjugate, another cytoxic agent, radiation or othertreatment administered simultaneously or in series. In exemplarytreatment method of the invention, an anti-FcRH5 antibody of theinvention is administered in combination with an anti-CD20 antibody,immunoglobulin, or CD20 binding fragment thereof, either together orsequentially. The anti-CD20 antibody may be a naked antibody or anantibody drug conjugate. In an embodiment of the combination therapy,the anti-FcRH5 antibody is an antibody of the present invention and theanti-CD20 antibody is Rituxan® (rituximab).

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology (3rd edition),A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd.,2000). See, in particular, the lists in FIGS. 11.57, 11.58 and 11.59.More specific examples include, but are not limited to, relapsed orrefractory NHL, front line low grade NHL, Stage III/IV NHL, chemotherapyresistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, smalllymphocytic lymphoma, B cell chronic lymphocytic leukemia and/orprolymphocytic leukemia and/or small lymphocytic lymphoma, B-cellprolymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic lymphoma,lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse large B-celllymphoma, aggressive NHL (including aggressive front-line NHL andaggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal large B-celllymphoma, primary effusion lymphoma, high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, Burkitt's lymphoma, precursor (peripheral) large granularlymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma.

Plasma cells disorders result from the uncontrolled division ormultiplication of a plasma cell clone. Plasma cells arise from activatedB lymphocytes (i.e., B cells). Each B cell produces a unique receptor,known as the B cell receptor, arrayed on its cell surface that isspecific for a foreign substance, i.e., antigen. When a B cell receptorbinds its cognate antigen, the cell expressing the receptor is activatedto re-enter the cell cycle, producing many clonal copies of itself. Theclones mature into plasma cells that reside principally in the bonemarrow and that are specialized to produce copies of the B cell receptorthat are released into the blood stream as antibodies. In a plasma celldisorder, the plasma cell or the parent B cell suffers genetic damageresulting in suppression of or insensitivity to the normal restraints oncell division and/or activity. Daughter plasma cells derived from suchcells are malignant in that they may divide unchecked and/or generateexcess amount of the same immunoglobulin (antibody). Often theimmunoglobulin produced is incomplete or has an incorrect conformationthat can result in accumulation of the protein (also known as monoclonalprotein, M protein, paraprotein or amyloid protein, dependent on thespecific disorder) in the serum, tissues or organs (especially thekidneys), leading to organ dysfunction and/or failure. Plasma celldisorders include monoclonal gammopathies of undetermined significance(MGUS), multiple myeloma (MM), macroglobulinemia, heavy chain diseases,and systemic light-chain amyloidosis (AL), which are differentiatedbased on the proliferative nature of the clone, the extent of marrowinvolvement, and the type of M protein expressed. Additional plasma celldisorders are solitary plasmacytoma, extramedullary plasmacytoma,multiple solitary plasmacytomas, plasma cell leukaemia, Waldenstrom'smacroglobulinaemia, B-cell non-Hodgkin lymphomas, B-cell chroniclymphocytic leukaemia. Although new immunotherapies such as rituximaband alemtuzumab have improved disease-free and overall survival in someB-cell malignancies, such therapies have not proven effective in thetreatment of plasma cell disorders in part because the target antigens,CD20 and CD52, respectively, are not sufficiently expressed by themalignant clonal plasma cells. Thus, there is a need for identificationand development of improved therapy of plasma cell disorders. (see U.S.Published Application Nos. 20080166742 and 20080317745, each of which isincorporate herein by reference in its entirety).

The expression of FcRH5 (IRTA2) on B cells, plasma cells, and multiplemyeloma cells (including cells from MM patient samples) has previouslybeen shown. (see U.S. Published Application No. 20060251662,incorporated herein by reference in its entirety). Accordingly, in lightof the FcRH5 expression pattern in multiple myeloma samples, themolecule is an excellent target for therapy of tumors in mammals,including plasma cell disorders, such as those described herein (i.e.multiple myeloma) and diseases associated with antibody-secretion, suchas allergy or autoimmune diseases.

A “disorder” is any condition that would benefit from treatment with asubstance/molecule or method of the invention. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include cancerous conditionssuch as malignant and benign tumors; non-leukemias and lymphoidmalignancies; neuronal, glial, astrocytal, hypothalamic and otherglandular, macrophagal, epithelial, stromal and blastocoelic disorders;and inflammatory, immunologic and other angiogenesis-related disorders.Disorders further include cancerous conditions such as B cellproliferative disorders and/or B cell tumors, e.g., lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or organs or a co-segregateor manifestation thereof or resulting condition therefrom. In many ofthese autoimmune and inflammatory disorders, a number of clinical andlaboratory markers may exist, including, but not limited to,hypergammaglobulinemia, high levels of autoantibodies, antigen-antibodycomplex deposits in tissues, benefit from corticosteroid orimmunosuppressive treatments, and lymphoid cell aggregates in affectedtissues. Without being limited to any one theory regarding B-cellmediated autoimmune disease, it is believed that B cells demonstrate apathogenic effect in human autoimmune diseases through a multitude ofmechanistic pathways, including autoantibody production, immune complexformation, dendritic and T-cell activation, cytokine synthesis, directchemokine release, and providing a nidus for ectopic neo-lymphogenesis.Each of these pathways may participate to different degrees in thepathology of autoimmune diseases.

“Autoimmune disease” can be an organ-specific disease (i.e., the immuneresponse is specifically directed against an organ system such as theendocrine system, the hematopoietic system, the skin, thecardiopulmonary system, the gastrointestinal and liver systems, therenal system, the thyroid, the ears, the neuromuscular system, thecentral nervous system, etc.) or a systemic disease which can affectmultiple organ systems (for example, systemic lupus erythematosus (SLE),rheumatoid arthritis, polymyositis, etc.). Preferred such diseasesinclude autoimmune rheumatologic disorders (such as, for example,rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLEand lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),autoimmune gastrointestinal and liver disorders (such as, for example,inflammatory bowel diseases (e.g., ulcerative colitis and Crohn'sdisease), autoimmune gastritis and pernicious anemia, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,and celiac disease), vasculitis (such as, for example, ANCA-negativevasculitis and ANCA-associated vasculitis, including Churg-Straussvasculitis, Wegener's granulomatosis, and microscopic polyangiitis),autoimmune neurological disorders (such as, for example, multiplesclerosis, opsoclonus myoclonus syndrome, myasthenia gravis,neuromyelitis optica, Parkinson's disease, Alzheimer's disease, andautoimmune polyneuropathies), renal disorders (such as, for example,glomerulonephritis, Goodpasture's syndrome, and Berger's disease),autoimmune dermatologic disorders (such as, for example, psoriasis,urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneouslupus erythematosus), hematologic disorders (such as, for example,thrombocytopenic purpura, thrombotic thrombocytopenic purpura,post-transfusion purpura, and autoimmune hemolytic anemia),atherosclerosis, uveitis, autoimmune hearing diseases (such as, forexample, inner ear disease and hearing loss), Behcet's disease,Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders(such as, for example, diabetic-related autoimmune diseases such asinsulin-dependent diabetes mellitus (IDDM), Addison's disease, andautoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

Specific examples of other autoimmune diseases as defined herein, whichin some cases encompass those listed above, include, but are not limitedto, arthritis (acute and chronic, rheumatoid arthritis includingjuvenile-onset rheumatoid arthritis and stages such as rheumatoidsynovitis, gout or gouty arthritis, acute immunological arthritis,chronic inflammatory arthritis, degenerative arthritis, type IIcollagen-induced arthritis, infectious arthritis, Lyme arthritis,proliferative arthritis, psoriatic arthritis, Still's disease, vertebralarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis,menopausal arthritis, estrogen-depletion arthritis, and ankylosingspondylitis/rheumatoid spondylitis), autoimmune lymphoproliferativedisease, inflammatory hyperproliferative skin diseases, psoriasis suchas plaque psoriasis, gutatte psoriasis, pustular psoriasis, andpsoriasis of the nails, atopy including atopic diseases such as hayfever and Job's syndrome, dermatitis including contact dermatitis,chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis,allergic contact dermatitis, hives, dermatitis herpetiformis, nummulardermatitis, seborrheic dermatitis, non-specific dermatitis, primaryirritant contact dermatitis, and atopic dermatitis, x-linked hyper IgMsyndrome, allergic intraocular inflammatory diseases, urticaria such aschronic allergic urticaria and chronic idiopathic urticaria, includingchronic autoimmune urticaria, myositis, polymyositis/dermatomyositis,juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma(including systemic scleroderma), sclerosis such as systemic sclerosis,multiple sclerosis (MS) such as spino-optical MS, primary progressive MS(PPMS), and relapsing remitting MS (RRMS), progressive systemicsclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata,ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, gastrointestinal inflammation, colitis suchas ulcerative colitis, colitis ulcerosa, microscopic colitis,collagenous colitis, colitis polyposa, necrotizing enterocolitis, andtransmural colitis, and autoimmune inflammatory bowel disease), bowelinflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosingcholangitis, respiratory distress syndrome, including adult or acuterespiratory distress syndrome (ARDS), meningitis, inflammation of all orpart of the uvea, iritis, choroiditis, an autoimmune hematologicaldisorder, graft-versus-host disease, angioedema such as hereditaryangioedema, cranial nerve damage as in meningitis, herpes gestationis,pemphigoid gestationis, pruritis scroti, autoimmune premature ovarianfailure, sudden hearing loss due to an autoimmune condition,IgE-mediated diseases such as anaphylaxis and allergic and atopicrhinitis, encephalitis such as Rasmussen's encephalitis and limbicand/or brainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressiveGN(RPGN), proliferative nephritis, autoimmune polyglandular endocrinefailure, balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, food allergies, drug allergies, insect allergies, rareallergic disorders such as mastocytosis, allergic reaction, eczemaincluding allergic or atopic eczema, asteatotic eczema, dyshidroticeczema, and vesicular palmoplantar eczema, asthma such as asthmabronchiale, bronchial asthma, and auto-immune asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,immune reactions against foreign antigens such as fetal A-B-O bloodgroups during pregnancy, chronic pulmonary inflammatory disease,autoimmune myocarditis, leukocyte adhesion deficiency, lupus, includinglupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus,extra-renal lupus, discoid lupus and discoid lupus erythematosus,alopecia lupus, SLE, such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatric IDDM,adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes,idiopathic diabetes insipidus, diabetic retinopathy, diabeticnephropathy, diabetic colitis, diabetic large-artery disorder, immuneresponses associated with acute and delayed hypersensitivity mediated bycytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosisincluding lymphomatoid granulomatosis, agranulocytosis, vasculitides(including large-vessel vasculitis such as polymyalgia rheumatica andgiant-cell (Takayasu's) arteritis, medium-vessel vasculitis such asKawasaki's disease and polyarteritis nodosa/periarteritis nodosa,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as fibrinoid necrotizingvasculitis and systemic necrotizing vasculitis, ANCA-negativevasculitis, and ANCA-associated vasculitis such as Churg-Strausssyndrome (CSS), Wegener's granulomatosis, and microscopic polyangiitis),temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombspositive anemia, Diamond Blackfan anemia, hemolytic anemia or immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia (anemia perniciosa), Addison's disease, pure red cellanemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia(s), cytopenias such as pancytopenia, leukopenia,diseases involving leukocyte diapedesis, CNS inflammatory disorders,Alzheimer's disease, Parkinson's disease, multiple organ injury syndromesuch as those secondary to septicemia, trauma or hemorrhage,antigen-antibody complex-mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, motoneuritis,allergic neuritis, Behçcet's disease/syndrome, Castleman's syndrome,Goodpasture's syndrome, Reynaud's syndrome, Sjögren's syndrome,Stevens-Johnson syndrome, pemphigoid or pemphigus such as pemphigoidbullous, cicatricial (mucous membrane) pemphigoid, skin pemphigoid,pemphigus vulgaris, paraneoplastic pemphigus, pemphigus foliaceus,pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus,epidermolysis bullosa acquisita, ocular inflammation, preferablyallergic ocular inflammation such as allergic conjunctivis, linear IgAbullous disease, autoimmune-induced conjunctival inflammation,autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermalinjury due to an autoimmune condition, preeclampsia, an immune complexdisorder such as immune complex nephritis, antibody-mediated nephritis,neuroinflammatory disorders, polyneuropathies, chronic neuropathy suchas IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (asdeveloped by myocardial infarction patients, for example), includingthrombotic thrombocytopenic purpura (TTP), post-transfusion purpura(PTP), heparin-induced thrombocytopenia, and autoimmune orimmune-mediated thrombocytopenia including, for example, idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, Grave's eye disease (ophthalmopathy orthyroid-associated ophthalmopathy), polyglandular syndromes such asautoimmune polyglandular syndromes, for example, type I (orpolyglandular endocrinopathy syndromes), paraneoplastic syndromes,including neurologic paraneoplastic syndromes such as Lambert-Eatonmyasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-personsyndrome, encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, pneumonitis such as lymphoidinterstitial pneumonitis (LIP), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré syndrome, Berger's disease (IgAnephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acutefebrile neutrophilic dermatosis, subcorneal pustular dermatosis,transient acantholytic dermatosis, cirrhosis such as primary biliarycirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiacor Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia such as mixed cryoglobulinemia,amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronaryartery disease, autoimmune ear disease such as autoimmune inner eardisease (AIED), autoimmune hearing loss, polychondritis such asrefractory or relapsed or relapsing polychondritis, pulmonary alveolarproteinosis, keratitis such as Cogan's syndrome/nonsyphiliticinterstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosaceaautoimmune, zoster-associated pain, amyloidosis, a non-cancerouslymphocytosis, a primary lymphocytosis, which includes monoclonal B celllymphocytosis (e.g., benign monoclonal gammopathy and monoclonalgammopathy of undetermined significance, MGUS), peripheral neuropathy,paraneoplastic syndrome, channelopathies such as epilepsy, migraine,arrhythmia, muscular disorders, deafness, blindness, periodic paralysis,and channelopathies of the CNS, autism, inflammatory myopathy, focal orsegmental or focal segmental glomerulosclerosis (FSGS), endocrineophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatologicaldisorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome,adrenalitis, gastric atrophy, presenile dementia, demyelinating diseasessuch as autoimmune demyelinating diseases and chronic inflammatorydemyelinating polyneuropathy, Dressler's syndrome, alopecia greata,alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon,esophageal dysmotility, sclerodactyl), and telangiectasia), male andfemale autoimmune infertility, e.g., due to anti-spermatozoanantibodies, mixed connective tissue disease, Chagas' disease, rheumaticfever, recurrent abortion, farmer's lung, erythema multiforme,post-cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung,allergic granulomatous angiitis, benign lymphocytic angiitis, Alport'ssyndrome, alveolitis such as allergic alveolitis and fibrosingalveolitis, interstitial lung disease, transfusion reaction, leprosy,malaria, parasitic diseases such as leishmaniasis, kypanosomiasis,schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan'ssyndrome, dengue, endocarditis, endomyocardial fibrosis, diffuseinterstitial pulmonary fibrosis, interstitial lung fibrosis, fibrosingmediastinitis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch'scyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV)infection, SCID, acquired immune deficiency syndrome (AIDS), echovirusinfection, sepsis (systemic inflammatory response syndrome (SIRS)),endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubellavirus infection, post-vaccination syndromes, congenital rubellainfection, Epstein-Barr virus infection, mumps, Evan's syndrome,autoimmune gonadal failure, Sydenham's chorea, post-streptococcalnephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,chorioiditis, giant-cell polymyalgia, chronic hypersensitivitypneumonitis, conjunctivitis, such as vernal catarrh,keratoconjunctivitis sicca, and epidemic keratoconjunctivitis,idiopathic nephritic syndrome, minimal change nephropathy, benignfamilial and ischemia-reperfusion injury, transplant organ reperfusion,retinal autoimmunity, joint inflammation, bronchitis, chronicobstructive airway/pulmonary disease, silicosis, aphthae, aphthousstomatitis, arteriosclerotic disorders (cerebral vascular insufficiency)such as arteriosclerotic encephalopathy and arterioscleroticretinopathy, aspermiogenese, autoimmune hemolysis, Boeck's disease,cryoglobulinemia, Dupuytren's contracture, endophthalmiaphacoanaphylactica, enteritis allergica, erythema nodosum leprosum,idiopathic facial paralysis, chronic fatigue syndrome, febrisrheumatica, Hamman-Rich's disease, sensoneural hearing loss,haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica (sympatheticophthalmitis), neonatal ophthalmitis, optic neuritis, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, lymphofollicular thymitis, vitiligo, toxic-shocksyndrome, food poisoning, conditions involving infiltration of T cells,leukocyte-adhesion deficiency, immune responses associated with acuteand delayed hypersensitivity mediated by cytokines and T-lymphocytes,diseases involving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, autoimmune polyendocrinopathies, oophoritis, primarymyxedema, autoimmune atrophic gastritis, rheumatic diseases, mixedconnective tissue disease, nephrotic syndrome, insulitis, polyendocrinefailure, autoimmune polyglandular syndromes, including polyglandularsyndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH),cardiomyopathy such as dilated cardiomyopathy, epidermolisis bullosaacquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome,primary sclerosing cholangitis, purulent or nonpurulent sinusitis, acuteor chronic sinusitis, ethmoid, frontal, maxillary, or sphenoidsinusitis, allergic sinusitis, an eosinophil-related disorder such aseosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgiasyndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropicalpulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, spondyloarthropathies,seronegative spondyloarthritides, polyendocrine autoimmune disease,sclerosing cholangitis, sclera, episclera, chronic mucocutaneouscandidiasis, Bruton's syndrome, transient hypogammaglobulinemia ofinfancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome,angiectasis, autoimmune disorders associated with collagen disease,rheumatism such as chronic arthrorheumatism, lymphadenitis, reduction inblood pressure response, vascular dysfunction, tissue injury,cardiovascular ischemia, hyperalgesia, renal ischemia, cerebralischemia, and disease accompanying vascularization, allergichypersensitivity disorders, glomerulonephritides, reperfusion injury,ischemic re-perfusion disorder, reperfusion injury of myocardial orother tissues, lymphomatous tracheobronchitis, inflammatory dermatoses,dermatoses with acute inflammatory components, multiple organ failure,bullous diseases, renal cortical necrosis, acute purulent meningitis orother central nervous system inflammatory disorders, ocular and orbitalinflammatory disorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, and endometriosis. Such diseases arecontemplated herein to be treated by the administration of an antibodywhich binds to a B cell surface marker, such as FcRH5, and includes theadministration of an unconjugated (“naked”) antibody or an antibodyconjugated to a cytotoxic agent as disclosed herein. Such diseases arealso contemplated herein to be treated by combination therapy includingan anti-FcRH5 antibody or anti-FcRH5 antibody drug conjugate of theinvention in combination with another antibody or antibody drugconjugate, another cytoxic agent, radiation or other treatmentadministered simultaneously or in series.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for a FcRH5 polypeptide-expressing cancer if, after receivinga therapeutic amount of an anti-FcRH5 antibody according to the methodsof the present invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of cancer cells or absence of the cancer cells; reduction inthe tumor size; inhibition (i.e., slow to some extent and preferablystop) of cancer cell infiltration into peripheral organs including thespread of cancer into soft tissue and bone; inhibition (i.e., slow tosome extent and preferably stop) of tumor metastasis; inhibition, tosome extent, of tumor growth; and/or relief to some extent, one or moreof the symptoms associated with the specific cancer; reduced morbidityand mortality, and improvement in quality of life issues. To the extentthe anti-FcRH5 antibody may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic. Reduction of these signsor symptoms may also be felt by the patient.

The above parameters for assessing successful treatment and improvementin the disease are readily measurable by routine procedures familiar toa physician. For cancer therapy, efficacy can be measured, for example,by assessing the time to disease progression (TTP) and/or determiningthe response rate (RR). Metastasis can be determined by staging testsand by bone scan and tests for calcium level and other enzymes todetermine spread to the bone. CT scans can also be done to look forspread to the pelvis and lymph nodes in the area. Chest X-rays andmeasurement of liver enzyme levels by known methods are used to look formetastasis to the lungs and liver, respectively. Other routine methodsfor monitoring the disease include transrectal ultrasonography (TRUS)and transrectal needle biopsy (TRNB).

For bladder cancer, which is a more localized cancer, methods todetermine progress of disease include urinary cytologic evaluation bycystoscopy, monitoring for presence of blood in the urine, visualizationof the urothelial tract by sonography or an intravenous pyelogram,computed tomography (CT) and magnetic resonance imaging (MRI). Thepresence of distant metastases can be assessed by CT of the abdomen,chest x-rays, or radionuclide imaging of the skeleton.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

An “individual” is a vertebrate. In certain embodiments, the vertebrateis a mammal. Mammals include, but are not limited to, farm animals (suchas cows), sport animals, pets (such as cats, dogs, and horses),primates, mice and rats. In certain embodiments, a mammal is a human.

“Mammal” for purposes of the treatment of, alleviating the symptoms of acancer refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

By “solid phase” or “solid support” is meant a non-aqueous matrix towhich an antibody of the present invention can adhere or attach.Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g., an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as an FcRH5 antibody) to a mammal. The components of the liposomeare commonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

A “small” molecule or “small” organic molecule is defined herein to havea molecular weight below about 500 Daltons.

An “individual,” “subject,” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. mammals include, but are notlimited to, farm animals (such as cows), sport animals, pets (such ascats, dogs, and horses), primates, mice and rats. In certainembodiments, a mammal is human.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulation may be sterile.

A “sterile” formulation is aseptic of free from all livingmicroorganisms and their spores.

An “effective amount” of an antibody as disclosed herein is an amountsufficient to carry out a specifically stated purpose. An “effectiveamount” may be determined empirically and in a routine manner, inrelation to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anantibody or other drug effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See the definition herein of“treating”. To the extent the drug may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

A “growth inhibitory amount” of an anti-FcRH5 antibody is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “growth inhibitory amount” ofan anti-FcRH5 antibody for purposes of inhibiting neoplastic cell growthmay be determined empirically and in a routine manner.

A “cytotoxic amount” of an anti-FcRH5 antibody is an amount capable ofcausing the destruction of a cell, especially tumor, e.g., cancer cell,either in vitro or in vivo. A “cytotoxic amount” of an anti-FcRH5antibody for purposes of inhibiting neoplastic cell growth may bedetermined empirically and in a routine manner.

A “FcRH5-expressing cell” is a cell which expresses an endogenous ortransfected FcRH5 polypeptide either on the cell surface or in asecreted form. A “FcRH5-expressing cancer” is a cancer comprising cellsthat have a FcRH5 polypeptide present on the cell surface or thatproduce and secrete a FcRH5 polypeptide. A “FcRH5-expressing cancer”optionally produces sufficient levels of FcRH5 polypeptide on thesurface of cells thereof, such that an anti-FcRH5 antibody can bindthereto and have a therapeutic effect with respect to the cancer. Inanother embodiment, a “FcRH5-expressing cancer” optionally produces andsecretes sufficient levels of FcRH5 polypeptide, such that an anti-FcRH5antibody antagonist can bind thereto and have a therapeutic effect withrespect to the cancer. With regard to the latter, the antagonist may bean antisense oligonucleotide which reduces, inhibits or preventsproduction and secretion of the secreted FcRH5 polypeptide by tumorcells. A cancer which “overexpresses” a FcRH5 polypeptide is one whichhas significantly higher levels of FcRH5 polypeptide at the cell surfacethereof, or produces and secretes, compared to a noncancerous cell ofthe same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. FcRH5polypeptide overexpression may be determined in a detection orprognostic assay by evaluating increased levels of the FcRH5 proteinpresent on the surface of a cell, or secreted by the cell (e.g., via animmunohistochemistry assay using anti-FcRH5 antibodies prepared againstan isolated FcRH5 polypeptide which may be prepared using recombinantDNA technology from an isolated nucleic acid encoding the FcRH5polypeptide; FACS analysis, etc.). Alternatively, or additionally, onemay measure levels of FcRH5 polypeptide-encoding nucleic acid or mRNA inthe cell, e.g., via fluorescent in situ hybridization using a nucleicacid based probe corresponding to a FcRH5-encoding nucleic acid or thecomplement thereof; (FISH; see WO98/45479 published October, 1998),Southern blotting, Northern blotting, or polymerase chain reaction (PCR)techniques, such as real time quantitative PCR (RT-PCR). One may alsostudy FcRH5 polypeptide overexpression by measuring shed antigen in abiological fluid such as serum, e.g., using antibody-based assays (seealso, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995;and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Aside from theabove assays, various in vivo assays are available to the skilledpractitioner. For example, one may expose cells within the body of thepatient to an antibody which is optionally labeled with a detectablelabel, e.g., a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g., by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkyatingagents, antimetabolites, spindle poison plant alkaloids,cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SARASAR™, SCH 66336,Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifamib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammall, calicheamicin omegaIl (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially aFcRH5-expressing cancer cell, either in vitro or in vivo. Thus, thegrowth inhibitory agent may be one which significantly reduces thepercentage of FcRH5-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess, such as proteolytic cleavage of a peptide linker of the ADC, orhydrolysis of a functional group such as a hydrazone, ester, or amide.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC) whereby the covalent attachment, i.e. linker, betweenthe drug moiety (D) and the antibody (Ab) is broken, resulting in thefree drug dissociated from the antibody inside the cell. The cleavedmoieties of the ADC are thus intracellular metabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, cytostatic orgrowth inhibitory effect of an ADC or an intracellular metabolite of anADC. Cytotoxic activity may be expressed as the IC₅₀ value, which is theconcentration (molar or mass) per unit volume at which half the cellssurvive.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (1-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen, phosphorusand sulfur, the remaining ring atoms being C, where one or more ringatoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocyclesare described in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring carbon atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.Heteroaryl groups are optionally substituted independently with one ormore substituents described herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR',—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drugto a mammal. The components of the liposome are commonly arranged in abilayer formation, similar to the lipid arrangement of biologicalmembranes.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, trifluoroacetic acid, maleic acid, succinicacid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalicacid, glycolic acid, salicylic acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha hydroxy acid, such ascitric acid or tartaric acid, an amino acid, such as aspartic acid orglutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid,a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid,or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater.

The term “protecting group” refers to a substituent that is commonlyemployed to block or protect a particular functionality while reactingother functional groups on the compound. For example, an“amino-protecting group” is a substituent attached to an amino groupthat blocks or protects the amino functionality in the compound.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and9-fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a “hydroxy-protectinggroup” refers to a substituent of a hydroxy group that blocks orprotects the hydroxy functionality. Suitable protecting groups includeacetyl and silyl. A “carboxy-protecting group” refers to a substituentof the carboxy group that blocks or protects the carboxy functionality.Common carboxy-protecting groups include phenylsulfonylethyl,cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(trimethylsilyl)ethoxymethyl,2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl,2-(diphenylphosphino)-ethyl, nitroethyl and the like. For a generaldescription of protecting groups and their use, see T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York,1991.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

Abbreviations

Linker Components:

MC=6-maleimidocaproyl

Val-Cit or “vc”=valine-citrulline (an exemplary dipeptide in a proteasecleavable linker)

Citrulline=2-amino-5-ureido pentanoic acid

PAB=p-aminobenzyloxycarbonyl (an example of a “self immolative” linkercomponent)

Me-Val-Cit=N-methyl-valine-citrulline (wherein the linker peptide bondhas been modified to prevent its cleavage by cathepsin B)

MC(PEG)6-OH=maleimidocaproyl-polyethylene glycol (can be attached toantibody cysteines).

Cytotoxic Drugs:

MMAE=mono-methyl auristatin E (MW 718)

MMAF=variant of auristatin E (MMAE) with a phenylalanine at theC-terminus of the drug (MW 731.5)

MMAF-DMAEA=MMAF with DMAEA (dimethylaminoethylamine) in an amide linkageto the C-terminal phenylalanine (MW 801.5)

MMAF-TEG=MMAF with tetraethylene glycol esterified to the phenylalanine

MMAF-NtBu=N-t-butyl, attached as an amide to C-terminus of MMAF

DM1=N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine

DM3=N(2′)-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine

DM4=N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine

Further abbreviations are as follows: AE is auristatin E, Boc isN-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4-dimethylaminopyridine, DME isethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF isN,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid),DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (is,2R)-(+)-norephedrine, PBS is phosphate-buffered saline (pH 7.4), PEG ispolyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as an intramolecular or intermoleculardisulfide bridge.

The term “thiol reactivity value” is a quantitative characterization ofthe reactivity of free cysteine amino acids. The thiol reactivity valueis the percentage of a free cysteine amino acid in a cysteine engineeredantibody which reacts with a thiol-reactive reagent, and converted to amaximum value of 1. For example, a free cysteine amino acid on acysteine engineered antibody which reacts in 100% yield with athiol-reactive reagent, such as a biotin-maleimide reagent, to form abiotin-labelled antibody has a thiol reactivity value of 1.0. Anothercysteine amino acid engineered into the same or different parentantibody which reacts in 80% yield with a thiol-reactive reagent has athiol reactivity value of 0.8. Another cysteine amino acid engineeredinto the same or different parent antibody which fails totally to reactwith a thiol-reactive reagent has a thiol reactivity value of 0.Determination of the thiol reactivity value of a particular cysteine maybe conducted by ELISA assay, mass spectroscopy, liquid chromatography,autoradiography, or other quantitative analytical tests.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody may be directed against a target antigenof interest, e.g. a biologically important polypeptide. Antibodiesdirected against nonpolypeptide antigens (such as tumor-associatedglycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated.

TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop−stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1, 1,0, 0,−6, 0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */{−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */ { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */{−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */ {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */{−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */{−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */ {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */ { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M, 0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1, 0,−2,0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0, 1,−5,2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4, 3}, /*R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6, 0,−1, 0,−2,2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0, 0,−3,−2, 1,_M,1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0, 0,−3, 0,−1, 0,0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U */ { 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /*V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2, 2,−2,_M,−1,−2,−2,−1, 0, 0,4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7, 0,−7,−3,−5,0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */ { 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /*Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1, 0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2,0, 0,10,−4}, /* Z */ { 0, 1,−5, 2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0,3, 0, 0, 0, 0,−2,−6, 0,−4, 4} }; /*  */ #include <stdio.h> #include<ctype.h> #define MAXJMP 16 /* max jumps in a diag */ #define MAXGAP 24/* don't continue to penalize gaps larger than this */ #define JMPS 1024/* max jmps in an path */ #define MX 4 /* save if there's at least MX−1bases since last jmp */ #define DMAT 3 /* value of matching bases */#define DMIS 0 /* penalty for mismatched bases */ #define DINS0 8 /*penalty for a gap */ #define DINS1 1 /* penalty per base */ #definePINS0 8 /* penalty for a gap */ #define PINS1 4 /* penalty per residue*/ struct jmp { short n[MAXJMP]; /* size of jmp (neg for dely) */unsigned short x[MAXJMP]; /* base no. of jmp in seq x */ }; /* limitsseq to 2{circumflex over ( )}16 −1 */ struct diag { int score; /* scoreat last jmp */ long offset; /* offset of prev block */ short ijmp; /*current jmp index */ struct jmp jp; /* list of jmps */ }; struct path {int spc; /* number of leading spaces */ short n[JMPS];/* size of jmp(gap) */ int x[JMPS];/* loc of jmp (last elem before gap) */ }; char*ofile; /* output file name */ char *namex[2]; /* seq names: getseqs( )*/ char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs:getseqs( ) */ int dmax; /* best diag: nw( ) */ int dmax0; /* final diag*/ int dna; /* set if dna: main( ) */ int endgaps; /* set if penalizingend gaps */ int gapx, gapy; /* total gaps in seqs */ int len0, len1; /*seq lens */ int ngapx, ngapy; /* total size of gaps */ int smax; /* maxscore: nw( ) */ int *xbm; /* bitmap for matching */ long offset; /*current offset in jmp file */ struct diag *dx; /* holds diagonals */struct path pp[2]; /* holds path for seqs */ char *calloc( ), *malloc(), *index( ), *strcpy( ); char *getseq( ), *g_calloc( ); /*Needleman-Wunsch alignment program  *  * usage: progs file1 file2 * where file1 and file2 are two dna or two protein sequences.  * Thesequences can be in upper- or lower-case an may contain ambiguity  * Anylines beginning with ‘;’, ‘>’ or ‘<’ are ignored  * Max file length is65535 (limited by unsigned short x in the jmp struct)  * A sequence with1/3 or more of its elements ACGTU is assumed to be DNA  * Output is inthe file “align.out”  *  * The program may create a tmp file in /tmp tohold info about traceback.  * Original version developed under BSD 4.3on a vax 8650  */ #include “nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’−‘A’))|(1<<(‘N’−‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’−‘A’))|(1<<(‘Q’−‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index*/ register *col0, *col1; /* score for curr,last row */ register xx, yy; /* index into seqs */ dx = (struct diag*)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely =(int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1 ; yy <= len1 ; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps ∥ ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >= dely[yy]){ dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else { dely[yy] −=ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >= dely[yy]) {dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else ndely[yy]++; }/* update penalty for del in y seq;  * favor new del over ongong del  */if (endgaps ∥ ndelx < MAXGAP) { if (col1[yy−1] − ins0 >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −= ins1; ndelx++; } }else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx = col1[yy−1] −(ins0+ins1); ndelx = 1; } else ndelx++; } /* pick the maximum score;we're favoring  * mis over any del and delx over dely  */ ...nw id = xx− yy + len1 − 1; if (mis >= delx && mis >= dely[yy]) col1[yy] = mis;else if (delx >= dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if(dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx > dx[id].jp.x[ij]+MX)∥ mis > dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx;dx[id].jp.x[ij] =xx; dx[id].score = delx; } else { col1[yy] = dely[yy];ij = dx[id].ijmp; if (dx[id].jp.n[0] && (!dna ∥ (ndely[yy] >= MAXJMP &&xx > dx[id].jp.x[ij]+MX) ∥ mis > dx[id].score+DINS0)) { dx[id].ijmp++;if (++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset= offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = −ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score =dely[yy]; } if (xx == len0 && yy < len1) { /* last col  */ if (endgaps)col1[yy] −= ins0+ins1*(len1−yy); if (col1[yy] > smax) { smax = col1[yy];dmax = id; } } } if (endgaps && xx < len0) col1[yy−1] −=ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax = col1[yy−1]; dmax =id; } tmp = col0; col0 = col1; col1 = tmp; } (void) free((char *)ndely);(void) free((char *)dely); (void) free((char *)col0); (void) free((char*)col1); } /*  *  * print( ) -- only routine visible outside this module *  * static:  * getmat( ) -- trace back best path, count matches:print( )  * pr_align( ) -- print alignment of described in array p[ ]:print( )  * dumpblock( ) -- dump a block of lines with numbers, stars:pr_align( )  * nums( ) -- put out a number line: dumpblock( )  *putline( ) -- put out a line (name, [num], seq, [num]): dumpblock( )  *stars( ) - -put a line of stars: dumpblock( )  * stripname( ) -- stripany path and prefix from a seqname  */ #include “nw.h” #define SPC 3#define P_LINE 256 /* maximum output line */ #define P_SPC 3 /* spacebetween name or num and seq */ extern _day[26][26]; int olen; /* setoutput line length */ FILE *fx; /* output file */ print( ) print { intlx, ly, firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, “w”)) ==0) { fprintf(stderr,“%s: can't write %s\n”, prog, ofile); cleanup(1); }fprintf(fx, “<first sequence: %s (length = %d)\n”, namex[0], len0);fprintf(fx, “<second sequence: %s (length = %d)\n”, namex[1], len1);olen = 60; lx = len0; ly = len1; firstgap = lastgap = 0; if (dmax < len1− 1) { /* leading gap in x */ pp[0].spc = firstgap = len1 − dmax − 1; ly−= pp[0].spc; } else if (dmax > len1 − 1) { /* leading gap in y */pp[1].spc = firstgap = dmax − (len1 − 1); lx −= pp[1].spc; } if (dmax0 <len0 − 1) { /* trailing gap in x */ lastgap = len0 − dmax0 −1; lx −=lastgap; } else if (dmax0 > len0 − 1) { /* trailing gap in y */ lastgap= dmax0 − (len0 − 1); ly −= lastgap; } getmat(lx, ly, firstgap,lastgap); pr_align( ); } /*  * trace back the best path, count matches */ static getmat(lx, ly, firstgap, lastgap) getmat int lx, ly; /*“core” (minus endgaps) */ int firstgap, lastgap; /* leading trailingoverlap */ { int nm, i0, i1, siz0, siz1; char outx[32]; double pct;register n0, n1; register char *p0, *p1; /* get total matches, score  */i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] + pp[1].spc; p1 = seqx[1] +pp[0].spc; n0 = pp[1].spc + 1; n1 = pp[0].spc + 1; nm = 0; while ( *p0&& *p1 ) { if (siz0) { p1++; n1++; siz0−−; } else if (siz1) { p0++;n0++; siz1−−; } else { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ ==pp[0].x[i0]) siz0 = pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 =pp[1].n[i1++]; p0++; p1++; } } /* pct homology:  * if penalizingendgaps, base is the shorter seq  * else, knock off overhangs and takeshorter core  */ if (endgaps) lx = (len0 < len1)? len0 : len1; else lx =(lx < ly)? lx : ly; pct = 100.*(double)nm/(double)lx; fprintf(fx, “\n”);fprintf(fx, “<%d match%s in an overlap of %d: %.2f percentsimilarity\n”, nm, (nm == 1)? “” : “es”, lx, pct); fprintf(fx, “<gaps infirst sequence: %d”, gapx); ...getmat if (gapx) { (void) sprintf(outx, “(%d %s%s)”, ngapx, (dna)? “base”:“residue”, (ngapx == 1)? “”:“s”);fprintf(fx,“%s”, outx); fprintf(fx, “, gaps in second sequence: %d”,gapy); if (gapy) { (void) sprintf(outx, “ (%d %s%s)”, ngapy, (dna)?“base”:“residue”, (ngapy == 1)? “”:“s”); fprintf(fx,“%s”, outx); } if(dna) fprintf(fx, “\n<score: %d (match = %d, mismatch = %d, gap penalty= %d + %d per base)\n”, smax, DMAT, DMIS, DINS0, DINS1); elsefprintf(fx, “\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %dper residue)\n”, smax, PINS0, PINS1); if (endgaps) fprintf(fx, “<endgapspenalized. left endgap: %d %s%s, right endgap: %d %s%s\n”, firstgap,(dna)? “base” : “residue”, (firstgap == 1)? “” : “s”, lastgap, (dna)?“base” : “residue”, (lastgap == 1)? “” : “s”); else fprintf(fx,“<endgaps not penalized\n”); }  static nm; /* matches in core -- forchecking */  static lmax; /* lengths of stripped file names */  staticij[2]; /* jmp index for a path */  static nc[2]; /* number at start ofcurrent line */  static ni[2]; /* current elem number -- for gapping */ static siz[2];  static char *ps[2]; /* ptr to current element */ static char *po[2]; /* ptr to next output char slot */  static charout[2][P_LINE]; /* output line */  static char star[P_LINE]; /* set bystars( ) */ /*  * print alignment of described in struct path pp[ ]  */static pr_align( ) pr_align { int nn; /* char count */ int more;register i; for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /*  *do we have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) { /* leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if(siz[i]) { /* in a gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen ∥ !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register i; for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ ∥ *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( ); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } /* * put out a number line: dumpblock( )  */ static nums(ix) nums int ix;/* index in out[ ] holding seq line */ { char nline[P_LINE]; register i,j; register char *pn, *px, *py; for (pn = nline, i = 0; i < lmax+P_SPC;i++, pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) {if (*py == ‘ ’ ∥ *py == ‘−’) *pn = ‘ ’; else { if (i%10 == 0 ∥ (i == 1&& nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j; j /= 10, px−−)*px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’; i++; } } *pn =‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx);(void) putc(‘\n’, fx); } /*  * put out a line (name, [num], seq, [num]):dumpblock( )  */ static putline(ix) putline int ix; { ...putline int i;register char *px; for (px = namex[ix], i = 0; *px && *px != ‘:’; px++,i++) (void) putc(*px, fx); for (; i < lmax+P_SPC; i++) (void) putc(‘ ’,fx); /* these count from 1:  * ni[ ] is current element (from 1)  * nc[] is number at start of current line  */ for (px = out[ix]; *px; px++)(void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /*  * put a line ofstars (seqs always in out[0], out[1]): dumpblock( )  */ static stars( )stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] ∥ (*out[0]== ‘ ’ && *(po[0]) == ‘ ’) ∥  !*out[1] ∥ (*out[1] == ‘ ’ && *(po[1]) ==‘ ’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,“r”)) == 0) {fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ ∥ *line ==‘<’ ∥ *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) ∥ islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s: malloc( ) failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ∥*line == ‘<’ ∥ *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1 */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP ∥ endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ∥endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw( )  */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

III. COMPOSITIONS AND METHODS OF THE INVENTION

The invention provides anti-FcRH5 antibodies or functional fragmentsthereof, and their method of use in the treatment of hematopoietictumors.

In one aspect, the invention provides an antibody which binds,preferably specifically, to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,antibody fragment, including Fab, Fab′, F(ab′)₂, and Fv fragment,diabody, single domain antibody, chimeric antibody, humanized antibody,single-chain antibody or antibody that competitively inhibits thebinding of an anti-FcRH5 polypeptide antibody to its respectiveantigenic epitope. Antibodies of the present invention may optionally beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, an auristatin, a maytansinoid, adolostatin derivative or a calicheamicin, an antibiotic, a radioactiveisotope, a nucleolytic enzyme, or the like. The antibodies of thepresent invention may optionally be produced in CHO cells or bacterialcells and preferably induce death of a cell to which they bind. Fordetection purposes, the antibodies of the present invention may bedetectably labeled, attached to a solid support, or the like.

In one aspect, the invention provides a humanized anti-FcRH5 antibodywherein the monovalent affinity of the antibody to FcRH5 (e.g. affinityof the antibody as a Fab fragment to FcRH5) is substantially the same asthe monovalent affinity of a murine antibody (e.g. affinity of themurine antibody as a Fab fragment to FcRH5) or a chimeric antibody (e.g.affinity of the chimeric antibody as a Fab fragment to FcRH5),comprising, consisting or consisting essentially of a light chain andheavy chain variable domain sequence as depicted in FIG. 9 (SEQ ID NO:18) and FIG. 10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the monovalent affinity of the antibody to FcRH5 (e.g.,affinity of the antibody as a Fab fragment to FcRH5) is lower, forexample at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60-fold lower, than themonovalent affinity of a murine antibody (e.g., affinity of the murineantibody as a Fab fragment to FcRH5) or a chimeric antibody (e.g.affinity of the chimeric antibody as a Fab fragment to FcRH5),comprising, consisting or consisting essentially of a light chain andheavy chain variable domain sequence as depicted in FIG. 9 (SEQ ID NO:18) and FIG. 10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the monovalent affinity of the antibody to FcRH5 (e.g.,affinity of the antibody as a Fab fragment to FcRH5) is greater, forexample at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold greater, than themonovalent affinity of a murine antibody (e.g., affinity of the murineantibody as a Fab fragment to FcRH5) or a chimeric antibody (e.g.affinity of the chimeric antibody as a Fab fragment to FcRH5),comprising, consisting or consisting essentially of a light chain andheavy chain variable domain sequence as depicted in FIG. 9 (SEQ ID NO:18) and FIG. 10 (SEQ ID NO: 20).

In one aspect, the invention provides a humanized anti-FcRH5 antibodywherein the affinity of the antibody in its bivalent form to FcRH5 (e.g.affinity of the antibody as an IgG to FcRH5) is substantially the sameas the affinity of a murine antibody (e.g. affinity of the antibody asan IgG to FcRH5) or a chimeric antibody (e.g. affinity of the chimericantibody as a Fab fragment to FcRH5) in its bivalent form, comprising,consisting or consisting essentially of a light chain and heavy chainvariable domain sequence as depicted in FIG. 9 (SEQ ID NO: 18) and FIG.10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g. affinity of the antibody as an IgG to FcRH5) is lower, forexample at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55 or 60-fold lower, as theaffinity of a murine antibody (e.g. affinity of the antibody as an IgGto FcRH5) or a chimeric antibody (e.g. affinity of the chimeric antibodyas an IgG fragment to FcRH5) in its bivalent form, comprising,consisting or consisting essentially of a light chain and heavy chainvariable domain sequence as depicted in FIG. 9 (SEQ ID NO: 18) and FIG.10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g. affinity of the antibody as an IgG to FcRH5) is greater, forexample at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold greater, than theaffinity of a murine antibody (e.g. affinity of the antibody as an IgGto FcRH5) or a chimeric antibody (e.g. affinity of the chimeric antibodyas an IgG fragment to FcRH5) in its bivalent form, comprising,consisting or consisting essentially of a light chain and heavy chainvariable domain sequence as depicted in FIG. 9 (SEQ ID NO: 18) and FIG.10 (SEQ ID NO: 20).

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.4 Nm. Ina further aspect, the invention provides a humanized anti-FcRH5 antibodywherein the affinity of the antibody in its bivalent form to FcRH5(e.g., affinity of the antibody as an IgG to FcRH5) is 0.4 nM+/−0.04.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.3 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.32 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.36 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.4 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.44 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.48 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.5 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is between 0.3nM and 0.5 nM. In another aspect, the invention provides a humanizedanti-FcRH5 antibody wherein the affinity of the antibody in its bivalentform to FcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) isbetween 0.32 nM and 0.48 nM.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is between0.36 nM and 0.44 nM.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.2 nM. Ina further aspect, the invention provides a humanized anti-FcRH5 antibodywherein the affinity of the antibody in its bivalent form to FcRH5(e.g., affinity of the antibody as an IgG to FcRH5) is 0.2 nM+/−0.02.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.1 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.12 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.14 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.16 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.18 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.2 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.22 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.24 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.26 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.28 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.30 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is between 0.1nM and 0.3 nM. In another aspect, the invention provides a humanizedanti-FcRH5 antibody wherein the affinity of the antibody in its bivalentform to FcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) isbetween 0.12 nM and 0.28 nM. In another aspect, the invention provides ahumanized anti-FcRH5 antibody wherein the affinity of the antibody inits bivalent form to FcRH5 (e.g., affinity of the antibody as an IgG toFcRH5) is between 0.14 nM and 0.26 nM. In another aspect, the inventionprovides a humanized anti-FcRH5 antibody wherein the affinity of theantibody in its bivalent form to FcRH5 (e.g., affinity of the antibodyas an IgG to FcRH5) is between 0.16 nM and 0.24 nM. In another aspect,the invention provides a humanized anti-FcRH5 antibody wherein theaffinity of the antibody in its bivalent form to FcRH5 (e.g., affinityof the antibody as an IgG to FcRH5) is between 0.18 nM and 0.22 nM.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.5 nM. Ina further aspect, the invention provides a humanized anti-FcRH5 antibodywherein the affinity of the antibody in its bivalent form to FcRH5(e.g., affinity of the antibody as an IgG to FcRH5) is 0.5 nM+/−0.1.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.4 nM orbetter.

In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.5 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.6 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is 0.7 nM orbetter. In another aspect, the invention provides a humanized anti-FcRH5antibody wherein the affinity of the antibody in its bivalent form toFcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) is between 0.3nM and 0.7 nM. In another aspect, the invention provides a humanizedanti-FcRH5 antibody wherein the affinity of the antibody in its bivalentform to FcRH5 (e.g., affinity of the antibody as an IgG to FcRH5) isbetween 0.4 nM and 0.6 nM. In another aspect, the invention provides ahumanized anti-FcRH5 antibody wherein the affinity of the antibody inits bivalent form to FcRH5 (e.g., affinity of the antibody as an IgG toFcRH5) is between 0.5 nM and 0.55 nM.

In one aspect, the monovalent affinity of the murine antibody to FcRH5is substantially the same as the binding affinity of a Fab fragmentcomprising variable domain sequences of FIG. 9 (SEQ ID NO: 18) and FIG.10 (SEQ ID NO: 20).

As is well-established in the art, binding affinity of a ligand to itsreceptor can be determined using any of a variety of assays, andexpressed in terms of a variety of quantitative values. Accordingly, inone embodiment, the binding affinity is expressed as Kd values andreflects intrinsic binding affinity (e.g., with minimized avidityeffects). Generally and preferably, binding affinity is measured invitro, whether in a cell-free or cell-associated setting. As describedin greater detail herein, fold difference in binding affinity can bequantified in terms of the ratio of the monovalent binding affinityvalue of a humanized antibody (e.g., in Fab form) and the monovalentbinding affinity value of a reference/comparator antibody (e.g., in Fabform) (e.g., a murine antibody having donor hypervariable regionsequences), wherein the binding affinity values are determined undersimilar assay conditions. Thus, in one embodiment, the fold differencein binding affinity is determined as the ratio of the Kd values of thehumanized antibody in Fab form and said reference/comparator Fabantibody. For example, in one embodiment, if an antibody of theinvention (A) has an affinity that is “3-fold lower” than the affinityof a reference antibody (M), then if the Kd value for A is 3×, the Kdvalue of M would be 1×, and the ratio of Kd of A to Kd of M would be3:1. Conversely, in one embodiment, if an antibody of the invention (C)has an affinity that is “3-fold greater” than the affinity of areference antibody (R), then if the Kd value for C is 1x, the Kd valueof R would be 3×, and the ratio of Kd of C to Kd of R would be 1:3. Anyof a number of assays known in the art, including those describedherein, can be used to obtain binding affinity measurements, including,for example, Biacore, radioimmunoassay (RIA) and ELISA.

In one aspect, an antibody that binds to FcRH5 is provided, wherein theantibody comprises:

(a) at least one, two, three, four, five or six HVRs selected from thegroup consisting of:

-   -   (i) HVR-L1 comprising the sequence KASQNVGSNVA (SEQ ID NO: 28)    -   (ii) HVR-L2 comprising the sequence SASYRYS (SEQ ID NO: 29)    -   (iii) HVR-L3 comprising the sequence QQYKTWT (SEQ ID NO: 30)    -   (iv) HVR-H1 comprising the sequence GYTFTNYGMN (SEQ ID NO: 37)    -   (v) HVR-H2 comprising the sequence NTYTGEPTYTDDFKG (SEQ ID NO:        38)    -   (vi) HVR-H3 comprising the sequence ARRSIPYYYAMDY (SEQ ID NO:        39)        In one embodiment, HVR-L1 of an antibody of the invention        comprises the sequence of SEQ ID NO: 28. In one embodiment,        HVR-L2 of an antibody of the invention comprises the sequence of        SEQ ID NO: 29 In one embodiment, HVR-L3 of an antibody of the        invention comprises the sequence of SEQ ID NO: 30. In one        embodiment, HVR-H1 of an antibody of the invention comprises the        sequence of SEQ ID NO: 37. In one embodiment, HVR-H2 of an        antibody of the invention comprises the sequence of SEQ ID        NO: 38. In one embodiment, HVR-H3 of an antibody of the        invention comprises the sequence of SEQ ID NO: 39. In one        embodiment, an antibody of the invention comprising these        sequences (in combination as described herein) is humanized or        human.

In one aspect, an antibody that binds to FcRH5 is provided, wherein theantibody comprises:

(a) at least one, two, three, four, five or six HVRs selected from thegroup consisting of:

-   -   (i) HVR-L1 comprising the sequence KASQNVGSNVA (SEQ ID NO: 28)    -   (ii) HVR-L2 comprising the sequence SASYRYS (SEQ ID NO: 29)    -   (iii) HVR-L3 comprising the sequence QQYKTWT (SEQ ID NO: 30)    -   (iv) HVR-H1 comprising the sequence GYTFTNYGMN (SEQ ID NO: 37)    -   (v) HVR-H2 comprising the sequence NTYTGEPTYTDDFKG (SEQ ID NO:        38)    -   (vi) HVR-H3 comprising the sequence ARRSIPYYYAMDY (SEQ ID NO:        39); and

(b) at least one variant HVR wherein the variant HVR sequence comprisesmodification of at least one residue of the sequence depicted in SEQ IDNOs: 28, 29, 30, 37, 38 or 39. In one embodiment, HVR-L1 of an antibodyof the invention comprises the sequence of SEQ ID NO: 28. In oneembodiment, HVR-L2 of an antibody of the invention comprises thesequence of SEQ ID NO: 29 In one embodiment, HVR-L3 of an antibody ofthe invention comprises the sequence of SEQ ID NO: 30. In oneembodiment, HVR-H1 of an antibody of the invention comprises thesequence of SEQ ID NO: 37. In one embodiment, HVR-H2 of an antibody ofthe invention comprises the sequence of SEQ ID NO: 38. In oneembodiment, HVR-H3 of an antibody of the invention comprises thesequence of SEQ ID NO: 39. In one embodiment, an antibody of theinvention comprising these sequences (in combination as describedherein) is humanized or human. In one embodiment, an antibody of theinvention comprising these sequences (in combination as describedherein) is humanized or human.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or six HVRs, wherein each HVR comprises, consists orconsists essentially of a sequence selected from the group consisting ofSEQ ID NOs: 28, 29, 30, 37, 38 or 39.

Variant HVRs in an antibody of an invention can have modifications ofone or more residues within the HVR.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or all of the HVR sequences depicted in FIGS. 9-12.

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides such an agent. For example, in oneembodiment, the invention provides a humanized antibody that elicitsand/or is expected to elicit a human anti-mouse antibody response (HAMA)at a substantially reduced level compared to an antibody comprising thesequence of SEQ ID NO: 18 & 20 in a host subject. In another example,the invention provides a humanized antibody that elicits and/or isexpected to elicit minimal or no human anti-mouse antibody response(HAMA). In one example, an antibody of the invention elicits anti-mouseantibody response that is at or less than a clinically-acceptable level.

A humanized antibody of the invention may comprise one or more humanand/or human consensus non-hypervariable region (e.g., framework)sequences in its heavy and/or light chain variable domain. In someembodiments, one or more additional modifications are present within thehuman and/or human consensus non-hypervariable region sequences. In oneembodiment, the heavy chain variable domain of an antibody of theinvention comprises a human consensus framework sequence, which in oneembodiment is the subgroup III consensus framework sequence. In oneembodiment, an antibody of the invention comprises a variant subgroupIII consensus framework sequence modified at least one amino acidposition. For example, in one embodiment, a variant subgroup IIIconsensus framework sequence may comprise a substitution at one or moreof positions 71, 73 and/or 78. In one embodiment, the light chainvariable domain of an antibody of the invention comprises a humanconsensus framework sequence, which in one embodiment is the κIconsensus framework sequence. In one embodiment, an antibody of theinvention comprises a variant κI consensus framework sequenced modifiedat least one amino acid position.

As is known in the art, and as described in greater detail herein below,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below). The invention provides antibodies comprisingmodifications in these hybrid hypervariable positions. In oneembodiment, these hypervariable positions include one or more positions26-30, 33-35B, 47-49, 57-65, 93, 94 and 101-102 in a heavy chainvariable domain. In one embodiment, these hybrid hypervariable positionsinclude one or more of positions 24-29, 35-36, 46-49, 56 and 97 in alight chain variable domain. In one embodiment, an antibody of theinvention comprises a human variant human subgroup consensus frameworksequence modified at one or more hybrid hypervariable positions.

An antibody of the invention can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In one embodiment, an antibody of the invention comprises atleast a portion (or all) of the framework sequence of human κ lightchain. In one embodiment, an antibody of the invention comprises atleast a portion (or all) of human κ subgroup I framework consensussequence.

In one aspect, an antibody of the invention is a humanized anti-FcRH5antibody conjugated to a cytotoxic agent. In one aspect, the humanizedanti-FcRH5 antibody conjugated to a cytotoxic agent inhibits tumorprogression in xenografts.

In one aspect, both the humanized antibody and chimeric antibody aremonovalent. In one embodiment, both the humanized and chimeric antibodycomprise a single Fab region linked to an Fc region. In one embodiment,the reference chimeric antibody comprises variable domain sequencesdepicted in FIG. 9 (SEQ ID NO: 18) and FIG. 10 (SEQ ID NO: 20)) linkedto a human Fc region. In one embodiment, the human Fc region is that ofan IgG (e.g., IgG1, 2, 3 or 4).

In one aspect, the antibodies of the invention include cysteineengineered antibodies where one or more amino acids of a parent antibodyare replaced with a free cysteine amino acid as disclosed inWO2006/034488; US 2007/0092940 (herein incorporated by reference in itsentirety). Any form of anti-FcRH5 antibody may be so engineered, i.e.mutated. For example, a parent Fab antibody fragment may be engineeredto form a cysteine engineered Fab, referred to herein as “ThioFab.”Similarly, a parent monoclonal antibody may be engineered to form a“ThioMab.” It should be noted that a single site mutation yields asingle engineered cysteine residue in a ThioFab, while a single sitemutation yields two engineered cysteine residues in a ThioMab, due tothe dimeric nature of the IgG antibody. The cysteine engineeredanti-FcRH5 antibodies of the invention include monoclonal antibodies,humanized or chimeric monoclonal antibodies, and antigen-bindingfragments of antibodies, fusion polypeptides and analogs thatpreferentially bind cell-associated FcRH5 polypeptides. A cysteineengineered antibody may alternatively comprise an antibody comprising acysteine at a position disclosed herein in the antibody or Fab,resulting from the sequence design and/or selection of the antibody,without necessarily altering a parent antibody, such as by phage displayantibody design and selection or through de novo design of light chainand/or heavy chain framework sequences and constant regions. A cysteineengineered antibody comprises one or more free cysteine amino acidshaving a thiol reactivity value in the ranges of 0.6 to 1.0; 0.7 to 1.0or 0.8 to 1.0. A free cysteine amino acid is a cysteine residue whichhas been engineered into the parent antibody and is not part of adisulfide bridge. Cysteine engineered antibodies are useful forattachment of cytotoxic and/or imaging compounds at the site of theengineered cysteine through, for example, a maleimide or haloacetyl. Thenucleophilic reactivity of the thiol functionality of a Cys residue to amaleimide group is about 1000 times higher compared to any other aminoacid functionality in a protein, such as amino group of lysine residuesor the N-terminal amino group. Thiol specific functionality iniodoacetyl and maleimide reagents may react with amine groups, buthigher pH (>9.0) and longer reaction times are required (Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press,London).

In an aspect, a cysteine engineered anti-FcRH5 antibody of the inventioncomprises an engineered cysteine at any suitable position, where theposition is numbered according to Kabat et al. in the light chain (seeKabat et al (1991) Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md.)and according to EU numbering in the heavy chain (including the Fcregion) (see Kabat et al. (1991), supra).

In a certain aspect, the invention concerns a cysteine engineeredanti-FcRH5 antibody, comprising an amino acid sequence having at leastabout 80% amino acid sequence identity, alternatively at least about81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to acysteine engineered antibody having a full-length amino acid sequence asdisclosed herein, or a cysteine engineered antibody amino acid sequencelacking the signal peptide as disclosed herein.

In a yet further aspect, the invention concerns an isolated cysteineengineered anti-FcRH5 antibody comprising an amino acid sequence that isencoded by a nucleotide sequence that hybridizes to the complement of aDNA molecule encoding (a) a cysteine engineered antibody having afull-length amino acid sequence as disclosed herein, (b) a cysteineengineered antibody amino acid sequence lacking the signal peptide asdisclosed herein, (c) an extracellular domain of a transmembranecysteine engineered antibody protein, with or without the signalpeptide, as disclosed herein, (d) an amino acid sequence encoded by anyof the nucleic acid sequences disclosed herein or (e) any otherspecifically defined fragment of a full-length cysteine engineeredantibody amino acid sequence as disclosed herein.

In a specific aspect, the invention provides an isolated cysteineengineered anti-FcRH5 antibody without the N-terminal signal sequenceand/or without the initiating methionine and is encoded by a nucleotidesequence that encodes such an amino acid sequence as described in.Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the cysteine engineered antibodyand recovering the cysteine engineered antibody from the cell culture.

Another aspect of the invention provides an isolated cysteine engineeredanti-FcRH5 antibody which is either transmembrane domain-deleted ortransmembrane domain-inactivated. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thecysteine engineered antibody and recovering the cysteine engineeredantibody from the cell culture.

In other aspects, the invention provides isolated anti-FcRH5 chimericcysteine engineered antibodies comprising any of the herein describedcysteine engineered antibody fused to a heterologous (non-FcRH5)polypeptide. Examples of such chimeric molecules comprise any of theherein described cysteine engineered antibodies fused to a heterologouspolypeptide such as, for example, an epitope tag sequence or a Fc regionof an immunoglobulin.

The cysteine engineered anti-FcRH5 antibody may be a monoclonalantibody, antibody fragment, chimeric antibody, humanized antibody,single-chain antibody or antibody that competitively inhibits thebinding of an anti-FcRH5 polypeptide antibody to its respectiveantigenic epitope. Antibodies of the present invention may optionally beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, an auristatin, a maytansinoid, adolostatin derivative or a calicheamicin, an antibiotic, a radioactiveisotope, a nucleolytic enzyme, or the like. The antibodies of thepresent invention may optionally be produced in CHO cells or bacterialcells and preferably inhibit the growth or proliferation of or inducethe death of a cell to which they bind. For diagnostic purposes, theantibodies of the present invention may be detectably labeled, attachedto a solid support, or the like.

In other aspects of the present invention, the invention providesvectors comprising DNA encoding any of the herein described anti-FcRH5antibodies and anti-FcRH5 cysteine engineered antibodies. Host cellscomprising any such vector are also provided. By way of example, thehost cells may be CHO cells, E. coli cells, or yeast cells. A processfor producing any of the herein described polypeptides is furtherprovided and comprises culturing host cells under conditions suitablefor expression of the desired polypeptide and recovering the desiredpolypeptide from the cell culture.

Cysteine engineered antibodies may be useful in the treatment of cancerand include antibodies specific for cell surface and transmembranereceptors, and tumor-associated antigens (TAA). Such antibodies may beused as naked antibodies (unconjugated to a drug or label moiety) or asantibody-drug conjugates (ADC). Cysteine engineered antibodies of theinvention may be site-specifically and efficiently coupled with athiol-reactive reagent. The thiol-reactive reagent may be amultifunctional linker reagent, a capture label reagent, a fluorophorereagent, or a drug-linker intermediate. The cysteine engineered antibodymay be labeled with a detectable label, immobilized on a solid phasesupport and/or conjugated with a drug moiety. Thiol reactivity may begeneralized to any antibody where substitution of amino acids withreactive cysteine amino acids may be made within the ranges in the lightchain selected from amino acid ranges: L10-L20, L105-L115, L109-L119,L116-L126, L122-L132, L163-L173, L200-L210; and within the ranges in theheavy chain selected from amino acid ranges: H1-H10, H18-H28, H79-H89,H107-H117, H109-H119, H111-H121, and in the Fc region within the rangesselected from H270-H280, H366-H376, H391-401, where the numbering ofamino acid positions begins at position 1 of the Kabat numbering system(Kabat et al. (1991) Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md.) and continues sequentially thereafter as disclosed in WO2006034488;US 2007/0092940. Thiol reactivity may also be generalized to certaindomains of an antibody, such as the light chain constant domain (CL) andheavy chain constant domains, CH1, CH2 and CH3. Cysteine replacementsresulting in thiol reactivity values of 0.6 and higher may be made inthe heavy chain constant domains α, δ, ε, γ, and μ of intact antibodies:IgA, IgD, IgE, IgG, and IgM, respectively, including the IgG subclasses:IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Such antibodies and their usesare disclosed in WO2006/034488; US 2007/0092940.

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, transmembrane proteins,signalling proteins, cell survival regulatory factors, cellproliferation regulatory factors, molecules associated with (for e.g.,known or suspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein, including but not limited toFcRH5). Cysteine engineered anti-FcRH5 antibodies of the inventionretain the antigen binding ability of their parent anti-FcRH5 antibodycounterparts. Thus, cysteine engineered anti-FcRH5 antibodies of theinvention are capable of binding, preferably specifically, to FcRH5antigens including human anti-FcRH5 isoforms beta and/or alpha,including when such antigens are expressed on the surface of cells,including, without limitation, B cells.

In one aspect, antibodies of the invention may be conjugated with anylabel moiety which can be covalently attached to the antibody through areactive moiety, an activated moiety, or a reactive cysteine thiol group(Singh et al (2002) Anal. Biochem. 304:147-15; Harlow E. and Lane, D.(1999) Using Antibodies: A Laboratory Manual, Cold Springs HarborLaboratory Press, Cold Spring Harbor, N.Y.; Lundblad R. L. (1991)Chemical Reagents for Protein Modification, 2nd ed. CRC Press, BocaRaton, Fla.). The attached label may function to: (i) provide adetectable signal; (ii) interact with a second label to modify thedetectable signal provided by the first or second label, e.g. to giveFRET (fluorescence resonance energy transfer); (iii) stabilizeinteractions or increase affinity of binding, with antigen or ligand;(iv) affect mobility, e.g. electrophoretic mobility orcell-permeability, by charge, hydrophobicity, shape, or other physicalparameters, or (v) provide a capture moiety, to modulate ligandaffinity, antibody/antigen binding, or ionic complexation.

Labelled cysteine engineered antibodies may be useful in diagnosticassays, e.g., for detecting expression of an antigen of interest inspecific cells, tissues, or serum. For diagnostic applications, theantibody will typically be labeled with a detectable moiety. Numerouslabels are available which can be generally grouped into the followingcategories:

Radioisotopes (radionuclides), such as ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S,⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu, ²¹¹At or²¹³Bi. Radioisotope labelled antibodies are useful in receptor targetedimaging experiments. The antibody can be labeled with ligand reagentsthat bind, chelate or otherwise complex a radioisotope metal where thereagent is reactive with the engineered cysteine thiol of the antibody,using the techniques described in Current Protocols in Immunology,Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, N.Y.,Pubs. (1991). Chelating ligands which may complex a metal ion includeDOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas, Tex.).Radionuclides can be targeted via complexation with the antibody-drugconjugates of the invention (Wu et al (2005) Nature Biotechnology 23(9):1137-1146).

Linker reagents such as DOTA-maleimide(4-maleimidobutyramidobenzyl-DOTA) can be prepared by the reaction ofaminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka) activated withisopropylchloroformate (Aldrich), following the procedure of Axworthy etal (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807). DOTA-maleimidereagents react with the free cysteine amino acids of the cysteineengineered antibodies and provide a metal complexing ligand on theantibody (Lewis et al (1998) Bioconj. Chem. 9:72-86). Chelating linkerlabelling reagents such as DOTA-NHS(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) are commercially available (Macrocyclics,Dallas, Tex.). Receptor target imaging with radionuclide labelledantibodies can provide a marker of pathway activation by detection andquantitation of progressive accumulation of antibodies in tumor tissue(Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210). Theconjugated radio-metals may remain intracellular following lysosomaldegradation.

Metal-chelate complexes suitable as antibody labels for imagingexperiments are disclosed: U.S. Pat. No. 5,342,606; U.S. Pat. No.5,428,155; U.S. Pat. No. 5,316,757; U.S. Pat. No. 5,480,990; U.S. Pat.No. 5,462,725; U.S. Pat. No. 5,428,139; U.S. Pat. No. 5,385,893; U.S.Pat. No. 5,739,294; U.S. Pat. No. 5,750,660; U.S. Pat. No. 5,834,456;Hnatowich et al (1983) J. Immunol. Methods 65:147-157; Meares et al(1984) Anal. Biochem. 142:68-78; Mirzadeh et al (1990) BioconjugateChem. 1:59-65; Meares et al (1990) J. Cancer 1990, Suppl. 10:21-26;Izard et al (1992) Bioconjugate Chem. 3:346-350; Nikula et al (1995)Nucl. Med. Biol. 22:387-90; Camera et al (1993) Nucl. Med. Biol.20:955-62; Kukis et al (1998) J. Nucl. Med. 39:2105-2110; Verel et al(2003) J. Nucl. Med. 44:1663-1670; Camera et al (1994) J. Nucl. Med.21:640-646; Ruegg et al Cancer Res. 50:4221-4226; Verel et al (2003) J.Nucl. Med. 44:1663-1670; Lee et al (2001) Cancer Res. 61:4474-4482;Mitchell, et al (2003) J. Nucl. Med. 44:1105-1112; Kobayashi et al(1999) Bioconjugate Chem. 10:103-111; Miederer et al (2004) J. Nucl.Med. 45:129-137; DeNardo et al (1998) Clinical Cancer Research4:2483-90; Blend et al (2003) Cancer Biotherapy & Radiopharmaceuticals18:355-363; Nikula et al (1999) J. Nucl. Med. 40:166-76; Kobayashi et al(1998) J. Nucl. Med. 39:829-36; Mardirossian et al (1993) Nucl. Med.Biol. 20:65-74; Roselli et al (1999) Cancer Biotherapy &Radiopharmaceuticals, 14:209-20.

Fluorescent labels such as rare earth chelates (europium chelates),fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansyl; Lissamine;cyanines; phycoerythrins; Texas Red; and analogs thereof. Thefluorescent labels can be conjugated to antibodies using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescent dyes and fluorescent label reagents include those which arecommercially available from Invitrogen/Molecular Probes (Eugene, Oreg.)and Pierce Biotechnology, Inc. (Rockford, Ill.).

Various enzyme-substrate labels are available or disclosed (U.S. Pat.No. 4,275,149). The enzyme generally catalyzes a chemical alteration ofa chromogenic substrate that can be measured using various techniques.For example, the enzyme may catalyze a color change in a substrate,which can be measured spectrophotometrically. Alternatively, the enzymemay alter the fluorescence or chemiluminescence of the substrate.Techniques for quantifying a change in fluorescence are described above.The chemiluminescent substrate becomes electronically excited by achemical reaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase(AP), β-galactosidase, glucoamylase, lysozyme, saccharide oxidases(e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al(1981) “Methods for the Preparation of Enzyme-Antibody Conjugates foruse in Enzyme Immunoassay”, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic Press, New York, 73:147-166.

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review, see U.S. Pat. No. 4,275,149and U.S. Pat. No. 4,318,980.

A label may be indirectly conjugated with an amino acid side chain, anactivated amino acid side chain, a cysteine engineered antibody, and thelike. For example, the antibody can be conjugated with biotin and any ofthe three broad categories of labels mentioned above can be conjugatedwith avidin or streptavidin, or vice versa. Biotin binds selectively tostreptavidin and thus, the label can be conjugated with the antibody inthis indirect manner. Alternatively, to achieve indirect conjugation ofthe label with the polypeptide variant, the polypeptide variant isconjugated with a small hapten (e.g., digoxin) and one of the differenttypes of labels mentioned above is conjugated with an anti-haptenpolypeptide variant (e.g., anti-digoxin antibody). Thus, indirectconjugation of the label with the polypeptide variant can be achieved(Hermanson, G. (1996) in Bioconjugate Techniques Academic Press, SanDiego).

The antibody of the present invention may be employed in any known assaymethod, such as ELISA, competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, (1987) MonoclonalAntibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.).

A detection label may be useful for localizing, visualizing, andquantitating a binding or recognition event. The labelled antibodies ofthe invention can detect cell-surface receptors. Another use fordetectably labelled antibodies is a method of bead-based immunocapturecomprising conjugating a bead with a fluorescent labelled antibody anddetecting a fluorescence signal upon binding of a ligand. Similarbinding detection methodologies utilize the surface plasmon resonance(SPR) effect to measure and detect antibody-antigen interactions.

Detection labels such as fluorescent dyes and chemiluminescent dyes(Briggs et al (1997) “Synthesis of Functionalised Fluorescent Dyes andTheir Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans.1:1051-1058) provide a detectable signal and are generally applicablefor labelling antibodies, preferably with the following properties: (i)the labelled antibody should produce a very high signal with lowbackground so that small quantities of antibodies can be sensitivelydetected in both cell-free and cell-based assays; and (ii) the labelledantibody should be photostable so that the fluorescent signal may beobserved, monitored and recorded without significant photo bleaching.For applications involving cell surface binding of labelled antibody tomembranes or cell surfaces, especially live cells, the labels preferably(iii) have good water-solubility to achieve effective conjugateconcentration and detection sensitivity and (iv) are non-toxic to livingcells so as not to disrupt the normal metabolic processes of the cellsor cause premature cell death.

Direct quantification of cellular fluorescence intensity and enumerationof fluorescently labelled events, e.g. cell surface binding ofpeptide-dye conjugates may be conducted on an system (FMAT® 8100 HTSSystem, Applied Biosystems, Foster City, Calif.) that automatesmix-and-read, non-radioactive assays with live cells or beads (Miraglia,“Homogeneous cell- and bead-based assays for high throughput screeningusing fluorometric microvolume assay technology”, (1999) J. ofBiomolecular Screening 4:193-204). Uses of labelled antibodies alsoinclude cell surface receptor binding assays, immunocapture assays,fluorescence linked immunosorbent assays (FLISA), caspase-cleavage(Zheng, “Caspase-3 controls both cytoplasmic and nuclear eventsassociated with Fas-mediated apoptosis in vivo”, (1998) Proc. Natl.Acad. Sci. USA 95:618-23; U.S. Pat. No. 6,372,907), apoptosis (Vermes,“A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V” (1995) J. Immunol. Methods 184:39-51) andcytotoxicity assays. Fluorometric microvolume assay technology can beused to identify the up or down regulation by a molecule that istargeted to the cell surface (Swartzman, “A homogeneous and multiplexedimmunoassay for high-throughput screening using fluorometric microvolumeassay technology”, (1999) Anal. Biochem. 271:143-51).

Labelled antibodies of the invention are useful as imaging biomarkersand probes by the various methods and techniques of biomedical andmolecular imaging such as: (i) MRI (magnetic resonance imaging); (ii)MicroCT (computerized tomography); (iii) SPECT (single photon emissioncomputed tomography); (iv) PET (positron emission tomography) Chen et al(2004) Bioconjugate Chem. 15:41-49; (v) bioluminescence; (vi)fluorescence; and (vii) ultrasound Immunoscintigraphy is an imagingprocedure in which antibodies labeled with radioactive substances areadministered to an animal or human patient and a picture is taken ofsites in the body where the antibody localizes (U.S. Pat. No.6,528,624). Imaging biomarkers may be objectively measured and evaluatedas an indicator of normal biological processes, pathogenic processes, orpharmacological responses to a therapeutic intervention. Biomarkers maybe of several types: Type 0 are natural history markers of a disease andcorrelate longitudinally with known clinical indices, e.g. MRIassessment of synovial inflammation in rheumatoid arthritis; Type Imarkers capture the effect of an intervention in accordance with amechanism-of-action, even though the mechanism may not be associatedwith clinical outcome; Type II markers function as surrogate endpointswhere the change in, or signal from, the biomarker predicts a clinicalbenefit to “validate” the targeted response, such as measured boneerosion in rheumatoid arthritis by CT. Imaging biomarkers thus canprovide pharmacodynamic (PD) therapeutic information about: (i)expression of a target protein, (ii) binding of a therapeutic to thetarget protein, i.e. selectivity, and (iii) clearance and half-lifepharmacokinetic data. Advantages of in vivo imaging biomarkers relativeto lab-based biomarkers include: non-invasive treatment, quantifiable,whole body assessment, repetitive dosing and assessment, i.e. multipletime points, and potentially transferable effects from preclinical(small animal) to clinical (human) results. For some applications,bioimaging supplants or minimizes the number of animal experiments inpreclinical studies.

Peptide labelling methods are well known. See Haugland, 2003, MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997)Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Glazer et al (1975) ChemicalModification of Proteins. Laboratory Techniques in Biochemistry andMolecular Biology (T. S. Work and E. Work, Eds.) American ElsevierPublishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984)Chemical Reagents for Protein Modification, Vols. I and II, CRC Press,New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”,Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter,Berlin and New York; and Wong (1991) Chemistry of Protein Conjugationand Cross-linking, CRC Press, Boca Raton, Fla.); De Leon-Rodriguez et al(2004) Chem. Eur. J. 10:1149-1155; Lewis et al (2001) Bioconjugate Chem.12:320-324; Li et al (2002) Bioconjugate Chem. 13:110-115; Mier et al(2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labelled with two moieties, a fluorescent reporterand quencher in sufficient proximity undergo fluorescence resonanceenergy transfer (FRET). Reporter groups are typically fluorescent dyesthat are excited by light at a certain wavelength and transfer energy toan acceptor, or quencher, group, with the appropriate Stokes shift foremission at maximal brightness. Fluorescent dyes include molecules withextended aromaticity, such as fluorescein and rhodamine, and theirderivatives. The fluorescent reporter may be partially or significantlyquenched by the quencher moiety in an intact peptide. Upon cleavage ofthe peptide by a peptidase or protease, a detectable increase influorescence may be measured (Knight, C. (1995) “Fluorimetric Assays ofProteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

The labelled antibodies of the invention may also be used as an affinitypurification agent. In this process, the labelled antibody isimmobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized antibody iscontacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the antigen from thepolypeptide variant.

Labelling reagents typically bear reactive functionality which may react(i) directly with a cysteine thiol of a cysteine engineered antibody toform the labelled antibody, (ii) with a linker reagent to form alinker-label intermediate, or (iii) with a linker antibody to form thelabelled antibody. Reactive functionality of labelling reagents include:maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g. NHS,N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride,2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite,although other functional groups can also be used.

An exemplary reactive functional group is N-hydroxysuccinimidyl ester(NHS) of a carboxyl group substituent of a detectable label, e.g. biotinor a fluorescent dye. The NHS ester of the label may be preformed,isolated, purified, and/or characterized, or it may be formed in situand reacted with a nucleophilic group of an antibody. Typically, thecarboxyl form of the label is activated by reacting with somecombination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O-(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the label. Insome cases, the label and the antibody may be coupled by in situactivation of the label and reaction with the antibody to form thelabel-antibody conjugate in one step. Other activating and couplingreagents include TBTU(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl sulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

Albumin Binding Peptide-Fab Compounds of the Invention:

In one aspect, the antibody of the invention is fused to an albuminbinding protein. Plasma-protein binding can be an effective means ofimproving the pharmacokinetic properties of short lived molecules.Albumin is the most abundant protein in plasma. Serum albumin bindingpeptides (ABP) can alter the pharmacodynamics of fused active domainproteins, including alteration of tissue uptake, penetration, anddiffusion. These pharmacodynamic parameters can be modulated by specificselection of the appropriate serum albumin binding peptide sequence (US20040001827). A series of albumin binding peptides were identified byphage display screening (Dennis et al. (2002) “Albumin Binding As AGeneral Strategy For Improving The Pharmacokinetics Of Proteins” J Biol.Chem. 277:35035-35043; WO 01/45746). Compounds of the invention includeABP sequences taught by: (i) Dennis et al (2002) J Biol. Chem.277:35035-35043 at Tables III and IV, page 35038; (ii) US 20040001827 at[0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 at pages 12-13, all ofwhich are incorporated herein by reference. Albumin Binding (ABP)-Fabsare engineered by fusing an albumin binding peptide to the C-terminus ofFab heavy chain in 1:1 stoichiometric ratio (1 ABP/1 Fab). It was shownthat association of these ABP-Fabs with albumin increased antibody halflife by more than 25 fold in rabbits and mice. The above describedreactive Cys residues can therefore be introduced in these ABP-Fabs andused for site-specific conjugation with cytotoxic drugs followed by invivo animal studies.

Exemplary albumin binding peptide sequences include, but are not limitedto the amino acid sequences listed in SEQ ID NOS: 47-51:

CDKTHTGGGSQRLMEDICLPRWGCLWEDDF SEQ ID NO: 47 QRLMEDICLPRWGCLWEDDFSEQ ID NO: 48 QRLIEDICLPRWGCLWEDDF SEQ ID NO: 49 RLIEDICLPRWGCLWEDDSEQ ID NO: 50 DICLPRWGCLW SEQ ID NO: 51

Antibody-Drug Conjugates

In another aspect, the invention provides immunoconjugates, orantibody-drug conjugates (ADC), comprising an antibody conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate). In another aspect, theinvention further provides methods of using the immunoconjugates. In oneaspect, an immunoconjugate comprises any of the above anti-FcRH5antibodies covalently attached to a cytotoxic agent or a detectableagent.

In one aspect, a FcRH5 antibody of the invention binds to the sameepitope on FcRH5 bound by another FcRH5 antibody. In another embodiment,a FcRH5 antibody of the invention binds to the same epitope on FcRH5bound by another FcRH5 antibody (i.e., commercially available anti-FcRH5antibodies).

In another aspect, a FcRH5 antibody of the invention binds to an epitopeon FcRH5 distinct from an epitope bound by another FcRH5 antibody. Inanother embodiment, a FcRH5 antibody of the invention binds to anepitope on FcRH5 distinct from an epitope on FcRH5 bound by anotherFcRH5 antibody (i.e., commercially available anti-FcRH5 antibodies).

In one aspect, an antibody of the invention specifically binds to FcRH5of a first animal species, and does not specifically bind to FcRH5 of asecond animal species. In one embodiment, the first animal species ishuman and/or primate (e.g., cynomolgus monkey), and the second animalspecies is murine (e.g., mouse) and/or canine. In one embodiment, thefirst animal species is human. In one embodiment, the first animalspecies is primate, for example cynomolgus monkey. In one embodiment,the second animal species is murine, for example mouse. In oneembodiment, the second animal species is canine.

In one aspect, the invention provides compositions comprising one ormore antibodies of the invention and a carrier. In one embodiment, thecarrier is pharmaceutically acceptable.

In one aspect, the invention provides nucleic acids encoding a FcRH5antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods for making an antibody ofthe invention. For example, the invention provides a method of making aFcRH5 antibody (which, as defined herein includes full length andfragments thereof), said method comprising expressing in a suitable hostcell a recombinant vector of the invention encoding said antibody (orfragment thereof), and recovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more FcRH5antibodies of the invention. In one embodiment, the compositioncomprises a nucleic acid of the invention. In one embodiment, acomposition comprising an antibody further comprises a carrier, which insome embodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (e.g., the antibody) to a subject.

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more FcRH5 antibodies of theinvention; and a second container comprising a buffer. In oneembodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antagonist antibody furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, a kit further comprises instructions foradministering the composition (e.g., the antibody) to a subject.

In one aspect, the invention provides use of a FcRH5 antibody of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor and/or acell proliferative disorder. In one embodiment, cancer, tumor and/orcell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor and/or acell proliferative disorder. In one embodiment, cancer, tumor and/orcell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor and/or acell proliferative disorder. In one embodiment, cancer, tumor and/orcell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor and/or acell proliferative disorder. In one embodiment, cancer, tumor and/orcell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumorand/or a cell proliferative disorder. In one embodiment, cancer, tumorand/or cell proliferative disorder is selected from lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor and/or a cellproliferative disorder. In one embodiment, cancer, tumor and/or cellproliferative disorder is selected from lymphoma, non-Hodgkins lymphoma(NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

In one aspect, the invention provides a method of inhibiting the growthof a cell that expresses FcRH5, said method comprising contacting saidcell with an antibody of the invention thereby causing an inhibition ofgrowth of said cell. In one embodiment, the antibody is conjugated to acytotoxic agent. In one embodiment, the antibody is conjugated to agrowth inhibitory agent.

In one aspect, the invention provides a method of therapeuticallytreating a mammal having a cancerous tumor comprising a cell thatexpresses FcRH5, said method comprising administering to said mammal atherapeutically effective amount of an antibody of the invention,thereby effectively treating said mammal. In one embodiment, theantibody is conjugated to a cytotoxic agent. In one embodiment, theantibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression of FcRH5, said method comprising administering to a subjectin need of such treatment an effective amount of an antibody of theinvention, thereby effectively treating or preventing said cellproliferative disorder. In one embodiment, said proliferative disorderis cancer. In one embodiment, the antibody is conjugated to a cytotoxicagent. In one embodiment, the antibody is conjugated to a growthinhibitory agent.

In one aspect, the invention provides a method for inhibiting the growthof a cell, wherein growth of said cell is at least in part dependentupon a growth potentiating effect of FcRH5, said method comprisingcontacting said cell with an effective amount of an antibody of theinvention, thereby inhibiting the growth of said cell. In oneembodiment, the antibody is conjugated to a cytotoxic agent. In oneembodiment, the antibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method of therapeuticallytreating a tumor in a mammal, wherein the growth of said tumor is atleast in part dependent upon a growth potentiating effect of FcRH5, saidmethod comprising contacting said cell with an effective amount of anantibody of the invention, thereby effectively treating said tumor. Inone embodiment, the antibody is conjugated to a cytotoxic agent. In oneembodiment, the antibody is conjugated to a growth inhibitory agent.

In one aspect, the invention provides a method of treating cancercomprising administering to a patient the pharmaceutical formulationcomprising an immunoconjugate described herein, acceptable diluent,carrier or excipient. In one embodiment, the cancer is selected from thelymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL) and mantle cell lymphoma. In one embodiment, the patientis administered a cytotoxic agent in combination with the antibody-drugconjugate compound.

In one aspect, the invention provides a method of inhibiting B cellproliferation comprising exposing a cell to an immunoconjugatecomprising an antibody of the invention under conditions permissive forbinding of the immunoconjugate to FcRH5. In one embodiment, the B cellproliferation is selected from lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL) and mantle cell lymphoma. In oneembodiment, the B cell is a xenograft. In one embodiment, the exposingtakes place in vitro. In one embodiment, the exposing taxes place invivo.

In one aspect, the invention provides a method of determining thepresence of FcRH5 in a sample suspected of containing FcRH5, said methodcomprising exposing said sample to an antibody of the invention, anddetermining binding of said antibody to FcRH5 in said sample whereinbinding of said antibody to FcRH5 in said sample is indicative of thepresence of said protein in said sample. In one embodiment, the sampleis a biological sample. In a further embodiment, the biological samplecomprises B cells. In one embodiment, the biological sample is from amammal experiencing or suspected of experiencing a B cell disorderand/or a B cell proliferative disorder including, but not limited to,lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL) and mantle cell lymphoma.

In one aspect, a method of diagnosing a cell proliferative disorderassociated with an increase in cells, such as B cells, expressing FcRH5is provided, the method comprising contacting a test cells in abiological sample with any of the above antibodies; determining thelevel of antibody bound to test cells in the sample by detecting bindingof the antibody to FcRH5; and comparing the level of antibody bound tocells in a control sample, wherein the level of antibody bound isnormalized to the number of FcRH5-expressing cells in the test andcontrol samples, and wherein a higher level of antibody bound in thetest sample as compared to the control sample indicates the presence ofa cell proliferative disorder associated with cells expressing FcRH5.

In one aspect, a method of detecting soluble FcRH5 in blood or serum,the method comprising contacting a test sample of blood or serum from amammal suspected of experiencing a B cell proliferative disorder with ananti-FcRH5 antibody of the invention and detecting a increase in solubleFcRH5 in the test sample relative to a control sample of blood or serumfrom a normal mammal. In an embodiment, the method of detecting isuseful as a method of diagnosing a B cell proliferative disorderassociated with an increase in soluble FcRH5 in blood or serum of amammal.

In one aspect, a method of binding an antibody of the invention to acell that expresses FcRH5, said method comprising contacting said cellwith an antibody of the invention. In one embodiment, the antibody isconjugated to a cytotoxic agent. In one embodiment, the antibody isconjugated to a growth inhibitory agent.

Methods of the invention can be used to affect any suitable pathologicalstate, for example, cells and/or tissues associated with expression ofFcRH5. In one embodiment, a cell that is targeted in a method of theinvention is a hematopoietic cell. For example, a hematopoietic cell canbe one selected from the group consisting of a lymphocyte, leukocyte,platelet, erythrocyte and natural killer cell. In one embodiment, a cellthat is targeted in a method of the invention is a B cell or T cell. Inone embodiment, a cell that is targeted in a method of the invention isa cancer cell. For example, a cancer cell can be one selected from thegroup consisting of a lymphoma cell, leukemia cell, or myeloma cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (e.g., a cancer cell) is exposedto radiation treatment or a chemotherapeutic agent.

In one aspect, the invention provides methods comprising administrationof an effective amount of an anti-FcRH5 antibody in combination with aneffective amount of another therapeutic agent (such as ananti-angiogenesis agent, another antibody, a chemotherapeutic agent, acytotoxic agent, an immunosuppressive agent, a prodrug, a cytokine,cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancervaccine, an analgesic, or a growth inhibitory agent). For example,anti-FcRH5 antibodies or immunoconjugates are used in combinations withan anti-cancer agent or an anti-angiogenic agent to treat variousneoplastic or non-neoplastic conditions. In particular examples, theanti-FcRH5 antibodies are used in combination with Velcade®(bortezomib), Revlimid® (lenalidomide), tamoxifen, letrozole,exemestane, anastrozole, irinotecan, cetuximab, fulvestrant,vinorelbine, bevacizumab, vincristine, cisplatin, gemcitabine,methotrexate, vinblastine, carboplatin, paclitaxel, docetaxel,pemetrexed, 5-fluorouracil, doxorubicin, bortezomib, lenalidomide,dexamethasone, melphalin, prednisone, vincristine, thalidomide.

Depending on the specific cancer indication to be treated, thecombination therapy of the invention can be combined with additionaltherapeutic agents, such as chemotherapeutic agents, or additionaltherapies such as radiotherapy or surgery. Many known chemotherapeuticagents can be used in the combination therapy of the invention.Preferably those chemotherapeutic agents that are standard for thetreatment of the specific indications will be used. Dosage or frequencyof each therapeutic agent to be used in the combination is preferablythe same as, or less than, the dosage or frequency of the correspondingagent when used without the other agent(s).

As described herein, FcRH5 (or IRTA2) expression has been observed to bederegulated in multiple myeloma and Burkitt lymphoma cell lines.Accordingly, in one embodiment of methods of the invention, a cell thatis targeted (e.g., a cancer cell) is one in which FcRH5 is expressed ascompared to a cell that does not express FcRH5. In a further embodiment,the targeted cell is a cancer cell in which FcRH5 expression is enhancedas compared to a normal non-cancer cell of the same tissue type. In oneembodiment, a method of the invention causes the death of a targetedcell.

In other aspects of the present invention, the invention providesvectors comprising DNA encoding any of the herein described antibodies.Host cell comprising any such vector are also provided. By way ofexample, the host cells may be CHO cells, E. coli cells, or yeast cells.A process for producing any of the herein described antibodies isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired antibody and recovering thedesired antibody from the cell culture.

In a still further aspect, the invention concerns a composition ofmatter comprising an anti-FcRH5 antibody as described herein, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another aspect of the present invention is directed to the use of ananti-FcRH5 polypeptide antibody as described herein, for the preparationof a medicament useful in the treatment of a condition which isresponsive to the anti-FcRH5 polypeptide antibody.

Another aspect of the invention is a composition comprising a mixture ofantibody-drug compounds of Formula I where the average drug loading perantibody is about 2 to about 5, or about 3 to about 4.

Another aspect of the invention is a pharmaceutical compositionincluding a Formula I ADC compound, a mixture of Formula I ADCcompounds, or a pharmaceutically acceptable salt or solvate thereof, anda pharmaceutically acceptable diluent, carrier, or excipient.

Another aspect provides a pharmaceutical combination comprising aFormula I ADC compound and a second compound having anticancerproperties or other therapeutic effects.

Another aspect is a method for killing or inhibiting the proliferationof tumor cells or cancer cells comprising treating the cells with anamount of an antibody-drug conjugate of Formula I, or a pharmaceuticallyacceptable salt or solvate thereof, being effective to kill or inhibitthe proliferation of the tumor cells or cancer cells.

Another aspect is a method of treating cancer comprising administeringto a patient a therapeutically effective amount of a pharmaceuticalcomposition including a Formula I ADC.

Another aspect includes articles of manufacture, i.e. kits, comprisingan antibody-drug conjugate, a container, and a package insert or labelindicating a treatment.

An aspect of the invention is a method for making a Formula I antibodydrug conjugate compound comprising the steps of: (a) reacting anengineered cysteine group of the cysteine engineered antibody with alinker reagent to form antibody-linker intermediate Ab-L; and (b)reacting Ab-L with an activated drug moiety D; whereby the antibody-drugconjugate is formed; or comprising the steps of: (c) reacting anucleophilic group of a drug moiety with a linker reagent to formdrug-linker intermediate D-L; and (d) reacting D-L with an engineeredcysteine group of the cysteine engineered antibody; whereby theantibody-drug conjugate is formed.

An aspect of the invention is an assay for detecting cancer cellscomprising: (a) exposing cells to a cysteine engineered anti-FcRH5antibody-drug conjugate; and (b) determining the extent of binding ofthe cysteine engineered anti-FcRH5 antibody-drug conjugate compound tothe cells.

A. Anti-FcRH5 Antibodies

In one embodiment, the present invention provides anti-FcRH5 antibodieswhich may find use herein as therapeutic agents. Exemplary antibodiesinclude polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

[Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR′N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g., by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and CO sequences for thehomologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, etal., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-FcRH5 antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. Reduction or eliminationof a HAMA response is a significant aspect of clinical development ofsuitable therapeutic agents. See, e.g., Khaxzaeli et al., J. Natl.Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986),41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J.Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988;Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature(1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495. Asdescribed herein, the invention provides antibodies that are humanizedsuch that HAMA response is reduced or eliminated. Variants of theseantibodies can further be obtained using routine methods known in theart, some of which are further described below. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), preferably the acceptor humanframeworks are from, or derived from, a human consensus frameworksequence as such frameworks have been demonstrated to have minimal, orno, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal; usually four, three, two or one amino acid differences onlyrelative to the human immunoglobulin sequence or consensus frameworksequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35B(H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

While “incorporation” of hypervariable region residues is discussedherein, it will be appreciated that this can be achieved in variousways, for example, nucleic acid encoding the desired amino acid sequencecan be generated by mutating nucleic acid encoding the mouse variabledomain sequence so that the framework residues thereof are changed toacceptor human framework residues, or by mutating nucleic acid encodingthe human variable domain sequence so that the hypervariable domainresidues are changed to non-human residues, or by synthesizing nucleicacid encoding the desired sequence, etc.

In the examples herein, hypervariable region-grafted variants weregenerated by Kunkel mutagenesis of nucleic acid encoding the humanacceptor sequences, using a separate oligonucleotide for eachhypervariable region. Kunkel et al., Methods Enzymol. 154:367-382(1987). Appropriate changes can be introduced within the frameworkand/or hypervariable region, using routine techniques, to correct andre-establish proper hypervariable region-antigen interactions.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins which bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants which can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, S., Proteins, 8:309(1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology,3:205 (1991)). In a monovalent phagemid display system, the gene fusionis expressed at low levels and wild type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S.Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have beenprepared in a number of ways including by altering a single gene byinserting random DNA sequences or by cloning a family of related genes.Methods for displaying antibodies or antigen binding fragments usingphage(mid) display have been described in U.S. Pat. Nos. 5,750,373,5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The library is then screened for expression of antibodies or antigenbinding proteins with the desired characteristics.

Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. For example, hypervariable region residues can be substitutedusing the Kunkel method. See, e.g., Kunkel et al., Methods Enzymol.154:367-382 (1987).

The sequence of oligonucleotides includes one or more of the designedcodon sets for the hypervariable region residues to be altered. A codonset is a set of different nucleotide triplet sequences used to encodedesired variant amino acids. Codon sets can be represented using symbolsto designate particular nucleotides or equimolar mixtures of nucleotidesas shown in below according to the IUB code.

IUB Codes

G Guanine

A Adenine

T Thymine

C Cytosine

R (A or G)

Y (C or T)

M (A or C)

K (G or T)

S(C or G)

W (A or T)

H (A or C or T)

B (C or G or T)

V (A or C or G)

D (A or G or T) H

N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al. Nucleic Acids Res.10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat'l. Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are either derivedfrom bacteriophage M13 vectors (the commercially available M13 mp 18 andM13 mp 19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedcan be inserted into one of these vectors in order to generatesingle-stranded template. Production of the single-stranded template isdescribed in sections 4.21-4.41 of Sambrook et al., above.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabelled with a 32-Phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: The singlestranded oligonucleotide is annealed to the single-stranded template asdescribed above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. Meth. Enzymol.,153:3 (1987). Thus, the DNA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

According to another method, antigen binding may be restored duringhumanization of antibodies through the selection of repairedhypervariable regions (See application Ser. No. 11/061,841, filed Feb.18, 2005). The method includes incorporating non-human hypervariableregions onto an acceptor framework and further introducing one or moreamino acid substitutions in one or more hypervariable regions withoutmodifying the acceptor framework sequence. Alternatively, theintroduction of one or more amino acid substitutions may be accompaniedby modifications in the acceptor framework sequence.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent accessible and highly diverse positions in ahypervariable region. As described above, a codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps can be isolated and cloned usingstandard recombinant techniques.

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of a humanized anti-FcRH5 antibody are contemplated. Forexample, the humanized antibody may be an antibody fragment, such as aFab, which is optionally conjugated with one or more cytotoxic agent(s)in order to generate an immunoconjugate. Alternatively, the humanizedantibody may be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; andU.S. Pat. No. 5,587,458. Fv and sFv are the only species with intactcombining sites that are devoid of constant regions; thus, they aresuitable for reduced nonspecific binding during in vivo use. sFv fusionproteins may be constructed to yield fusion of an effector protein ateither the amino or the carboxy terminus of an sFv. See AntibodyEngineering, ed. Borrebaeck, supra. The antibody fragment may also be a“linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 forexample. Such linear antibody fragments may be monospecific orbispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of a FcRH5 protein as described herein.Other such antibodies may combine a FcRH5 binding site with a bindingsite for another protein. Alternatively, an anti-FcRH5 arm may becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as tofocus and localize cellular defense mechanisms to the FcRH5-expressingcell. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express FcRH5. These antibodies possess aFcRH5-binding arm and an arm which binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fca antibody is shown in WO98/02463.U.S. Pat. Nos. 5,821,337 and 6,407,213 teach bispecificanti-ErbB2/anti-CD3 antibodies. Additional bispecific antibodies thatbind an epitope on the CD3 antigen and a second epitope have beendescribed. See, for example, U.S. Pat. No. 5,078,998 (anti-CD3/tumorcell antigen); U.S. Pat. No. 5,601,819 (anti-CD3/IL-2R; anti-CD3/CD28;anti-CD3/CD45); U.S. Pat. No. 6,129,914 (anti-CD3/malignant B cellantigen); U.S. Pat. No. 7,112,324 (anti-CD3/CD19); U.S. Pat. No.6,723,538 (anti-CD3/CCR5); U.S. Pat. No. 7,235,641 (anti-CD3/EpCAM);U.S. Pat. No. 7,262,276 (anti-CD3/ovarian tumor antigen); and U.S. Pat.No. 5,731,168 (anti-CD3/CD4IgG).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. Preferably, thefusion is with an Ig heavy chain constant domain, comprising at leastpart of the hinge, C_(H)2, and C_(H)3 regions. It is preferred to havethe first heavy-chain constant region (C_(H)1) containing the sitenecessary for light chain bonding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable host cell.This provides for greater flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yield of the desired bispecific antibody. It is,however, possible to insert the coding sequences for two or all threepolypeptide chains into a single expression vector when the expressionof at least two polypeptide chains in equal ratios results in highyields or when the ratios have no significant affect on the yield of thedesired chain combination.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.Bispecific antibodies produced in accordance with this approach arereferred to herein as “protuberance-into-cavity” antibodies.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise a V_(H)connected to a V_(L) by a linker which is too short to allow pairingbetween the two domains on the same chain. Accordingly, the V_(H) andV_(L) domains of one fragment are forced to pair with the complementaryV_(L) and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH₁-VH-CH₁-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

8. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

9. Immunoconjugates

The invention also pertains to immunoconjugates (interchangeablyreferred to as “antibody-drug conjugates,” or “ADCs”) comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

In certain embodiments, an immunoconjugate comprises an antibody and achemotherapeutic agent or other toxin. Chemotherapeutic agents useful inthe generation of such immunoconjugates have been described above.Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and ¹⁸⁶Re. Conjugates of the antibodyand cytotoxic agent are made using a variety of bifunctionalprotein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science, 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, auristatin peptides, such as monomethylauristatin(MMAE) (synthetic analog of dolastatin), maytansinoids, such as DM1, atrichothene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

Exemplary Immunoconjugates—Antibody-Drug Conjugates

An immunoconjugate (or “antibody-drug conjugate” (“ADC”)) of theinvention may be of Formula I, below, wherein an antibody is conjugated(i.e., covalently attached) to one or more drug moieties (D) through anoptional linker (L). ADCs may include thioMAb drug conjugates (“TDC”).Ab-(L-D)_(p)  I

Accordingly, the antibody may be conjugated to the drug either directlyor via a linker. In Formula I, p is the average number of drug moietiesper antibody, which can range, e.g., from about 1 to about 20 drugmoieties per antibody, and in certain embodiments, from 1 to about 8drug moieties per antibody. The invention includes a compositioncomprising a mixture of antibody-drug compounds of Formula I where theaverage drug loading per antibody is about 2 to about 5, or about 3 toabout 4.

a. Exemplary Linkers

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), and those resultingfrom conjugation with linker reagents: N-Succinimidyl 4-(2-pyridylthio)pentanoate forming linker moiety 4-mercaptopentanoic acid (“SPP”),N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate forminglinker moiety 4-((2,5-dioxopyrrolidin-1-yl)methylicyclohexanecarboxylicacid (“SMCC”, also referred to herein as “MCC”),2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl) butanoate forminglinker moiety 4-mercaptobutanoic acid (“SPDB”), N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”), ethyleneoxy —CH₂CH₂O— as one ormore repeating units (“EO” or “PEO”). Additional linker components areknown in the art and some are described herein. Various linkercomponents are known in the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug inthe cell. For example, an acid-labile linker (e.g., hydrazone),protease-sensitive (e.g., peptidase-sensitive) linker, photolabilelinker, dimethyl linker or disulfide-containing linker (Chari et al.,Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

In certain embodiments, a linker is as shown in the following FormulaII:-A_(a)-W_(w)—Y_(y)—  IIwherein A is a stretcher unit, and a is an integer from 0 to 1; W is anamino acid unit, and w is an integer from 0 to 12; Y is a spacer unit,and y is 0, 1, or 2; and Ab, D, and p are defined as above for FormulaI. Exemplary embodiments of such linkers are described in US2005-0238649 A1, which is expressly incorporated herein by reference.

In some embodiments, a linker component may comprise a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Exemplary stretcher units are shown below (wherein the wavy lineindicates sites of covalent attachment to an antibody):

In some embodiments, a linker component may comprise an amino acid unit.In one such embodiment, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes. See, e.g., Doronina et al. (2003) Nat. Biotechnol.21:778-784. Exemplary amino acid units include, but are not limited to,a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide. Exemplarydipeptides include: valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplarytripeptides include: glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally, as well as minor amino acidsand non-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

In some embodiments, a linker component may comprise a “spacer” unitthat links the antibody to a drug moiety, either directly or by way of astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon enzymatic (e.g., proteolytic) cleavage of theADC. Examples of non-self-immolative spacer units include, but are notlimited to, a glycine spacer unit and a glycine-glycine spacer unit.Other combinations of peptidic spacers susceptible to sequence-specificenzymatic cleavage are also contemplated. For example, enzymaticcleavage of an ADC containing a glycine-glycine spacer unit by atumor-cell associated protease would result in release of aglycine-glycine-drug moiety from the remainder of the ADC. In one suchembodiment, the glycine-glycine-drug moiety is then subjected to aseparate hydrolysis step in the tumor cell, thus cleaving theglycine-glycine spacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moietywithout a separate hydrolysis step. In certain embodiments, a spacerunit of a linker comprises a p-aminobenzyl unit. In one such embodiment,a p-aminobenzyl alcohol is attached to an amino acid unit via an amidebond, and a carbamate, methylcarbamate, or carbonate is made between thebenzyl alcohol and a cytotoxic agent. See, e.g., Hamann et al. (2005)Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment, thespacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments,the phenylene portion of a p-amino benzyl unit is substituted with Qm,wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4. Examples of self-immolative spacerunits further include, but are not limited to, aromatic compounds thatare electronically similar to p-aminobenzyl alcohol (see, e.g., US2005/0256030 A1), such as 2-aminoimidazol-5-methanol derivatives (Hay etal. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995,2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2]ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815); and2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem.,1990, 55, 5867). Elimination of amine-containing drugs that aresubstituted at the a-position of glycine (Kingsbury, et al., J. Med.Chem., 1984, 27, 1447) are also examples of self-immolative spacersuseful in ADCs.

In one embodiment, a spacer unit is a branched bis(hydroxymethyl)styrene(BHMS) unit as depicted below, which can be used to incorporate andrelease multiple drugs.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from1 to about 20.

In another embodiment, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the ADC. Thus, where a cysteine engineeredantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker.

Exemplary linker components and combinations thereof are shown below inthe context of ADCs of Formula II:

Linkers components, including stretcher, spacer, and amino acid units,may be synthesized by methods known in the art, such as those describedin US 2005-0238649 A1.

b. Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody conjugatedto one or more maytansinoid molecules. Maytansinoids are mitototicinhibitors which act by inhibiting tubulin polymerization. Maytansinewas first isolated from the east African shrub Maytenus serrata (U.S.Pat. No. 3,896,111). Subsequently, it was discovered that certainmicrobes also produce maytansinoids, such as maytansinol and C-3maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol andderivatives and analogues thereof are disclosed, for example, in U.S.Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946;4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;4,424,219; 4,450,254; 4,362,663; and 4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through disulfide and non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art and can be isolated from natural sources accordingto known methods or produced using genetic engineering and fermentationtechniques (U.S. Pat. No. 6,790,952; US 2005/0170475; Yu et al (2002)PNAS 99:7968-7973). Maytansinol and maytansinol analogues may also beprepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂₅₅);C-14-alkoxymethyl(demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Many positions on maytansine compounds are known to be useful as thelinkage position, depending upon the type of link. For example, forforming an ester linkage, the C-3 position having a hydroxyl group, theC-14 position modified with hydroxymethyl, the C-15 position modifiedwith a hydroxyl group and the C-20 position having a hydroxyl group areall suitable (U.S. Pat. No. 5,208,020; U.S. Pat. No. RE39151; U.S. Pat.No. 6,913,748; U.S. Pat. No. 7,368,565; US 2006/0167245; US2007/0037972).

Maytansinoid drug moieties include those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid drug moiety to a linker of an ADC. R mayindependently be H or a C₁-C₆ alkyl. The alkylene chain attaching theamide group to the sulfur atom may be methanyl, ethanyl, or propyl,i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020;U.S. Pat. No. 7,276,497; Chari et al (1992) Cancer Res. 52:127-131; Liuet al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe compounds of the invention, i.e. any combination of R and Sconfigurations at the chiral carbons of D. In one embodiment, themaytansinoid drug moiety will have the following stereochemistry:

Exemplary embodiments of maytansinoid drug moieities include: DM1; DM3;and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate. (WO2005/037992; US 2005/0276812 A1).

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations, (wherein Ab is antibody and p is 1 toabout 8):

In one embodiment, the antibody-drug conjugate is formed where DM4 islinked through an SPDB linker to a thiol group of the antibody (see U.S.Pat. Nos. 6,913,748 and 7,276,497 incorporated herein by reference intheir entirety).

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1, 2, 3, or 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in Erickson, et al(2006) Cancer Res. 66(8):4426-4433; U.S. Pat. Nos. 5,208,020, 5,416,064,US 2005/0276812 A1, and European Patent EP 0 425 235 B1, the disclosuresof which are hereby expressly incorporated by reference.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). Maytansinoids can besynthesized by known techniques or isolated from natural sources.Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.5,208,020 and in the other patents and nonpatent publications referredto hereinabove, such as maytansinol and maytansinol analogues modifiedin the aromatic ring or at other positions of the maytansinol molecule,such as various maytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1; Chari etal. Cancer Research 52:127-131 (1992); and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.Antibody-maytansinoid conjugates comprising the linker component SMCCmay be prepared as disclosed in US 2005/0276812 A1, “Antibody-drugconjugates and Methods.” The linkers comprise disulfide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups, or esterase labile groups, as disclosed in theabove-identified patents. Additional linkers are described andexemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). In certain embodiments, the couplingagent is N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) orN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In one embodiment, the linkage isformed at the C-3 position of maytansinol or a maytansinol analogue.

(2) Auristatins and Dolastatins

In some embodiments, an immunoconjugate comprises an antibody conjugatedto dolastatin or a dolastatin peptidic analog or derivative, e.g., anauristatin (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF (US 2005/0238649, disclosedin Senter et al, Proceedings of the American Association for CancerResearch, Volume 45, Abstract Number 623, presented Mar. 28, 2004, thedisclosure of which is expressly incorporated by reference in itsentirety).

A peptidic drug moiety may be selected from Formulas D_(E) and D_(F)below:

wherein the wavy line of D_(E) and D_(E) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COON, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(E) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006)Bioconjugate Chem. 17:114-124):

Other exemplary embodiments include monomethylvaline compounds havingphenylalanine carboxy modifications at the C-terminus of thepentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Other drug moieties include the following MMAF derivatives, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

In one aspect, hydrophilic groups including but not limited to,triethylene glycol esters (TEG), as shown above, can be attached to thedrug moiety at R¹¹. Without being bound by any particular theory, thehydrophilic groups assist in the internalization and non-agglomerationof the drug moiety.

Exemplary embodiments of ADCs of Formula I comprising anauristatin/dolastatin or derivative thereof are described in US2005-0238649 and Doronina et al. (2006) Bioconjugate Chem. 17:114-124,which is expressly incorporated herein by reference. Exemplaryembodiments of ADCs of Formula I comprising MMAE or MMAF and variouslinker components have the following structures and abbreviations(wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit” or “vc” is avaline-citrulline dipeptide; and “S” is a sulfur atom. It will be notedthat in certain of the structural descriptions of sulfur linked ADCherein the antibody is represented as “Ab-S” merely to indicate thesulfur link feature and not to indicate that a particular sulfur atombears multiple linker-drug moieties. The left parentheses of thefollowing structures may also be placed to the left of the sulfur atom,between Ab and S, which would be an equivalent description of the ADC ofthe invention described throughout herein.

Exemplary embodiments of ADCs of Formula I comprising MMAF and variouslinker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.Interestingly, immunoconjugates comprising MMAF attached to an antibodyby a linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker. See, Doronina etal. (2006) Bioconjugate Chem. 17:114-124. In such instances, drugrelease is believed to be effected by antibody degradation in the cell.Id. Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Liibke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. Auristatin/dolastatin drug moieties may beprepared according to the methods of: US 2005-0238649 A1; U.S. Pat. No.5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In particular, auristatin/dolastatin drug moieties of formula D_(F),such as MMAF and derivatives thereof, may be prepared using methodsdescribed in US 2005-0238649 A1 and Doronina et al. (2006) BioconjugateChem. 17:114-124. Auristatin/dolastatin drug moieties of formula D_(E),such as MMAE and derivatives thereof, may be prepared using methodsdescribed in Doronina et al. (2003) Nat. Biotech. 21:778-784.Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, andMC-vc-PAB-MMAE may be conveniently synthesized by routine methods, e.g.,as described in Doronina et al. (2003) Nat. Biotech. 21:778-784, andPatent Application Publication No. US 2005/0238649 A1, and thenconjugated to an antibody of interest.

(3) Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman etal., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998), and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug to which the antibody can beconjugated is QFA, which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents throughantibody-mediated internalization greatly enhances their cytotoxiceffects.

c. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to an antibody includeBCNU, streptozocin, vincristine and 5-fluorouracil, the family of agentsknown collectively as the LL-E33288 complex, described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. When the immunoconjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

In certain embodiments, an immunoconjugate may comprise an antibodyconjugated to a prodrug-activating enzyme that converts a prodrug (e.g.,a peptidyl chemotherapeutic agent, see WO 81/01145) to an active drug,such as an anti-cancer drug. Such immunoconjugates are useful inantibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymesthat may be conjugated to an antibody include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. Enzymes may be covalently bound to antibodies byrecombinant DNA techniques well known in the art. See, e.g., Neubergeret al., Nature 312:604-608 (1984).

d. Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis. Pharmaceutical formulations of Formula I antibody-drugconjugates may thus be a heterogeneous mixture of such conjugates withantibodies linked to 1, 2, 3, 4, or more drug moieties.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed,it has been shown that for certain ADCs, the optimal ratio of drugmoieties per antibody may be less than 8, and may be about 2 to about 5.See US 2005-0238649 A1.

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, and (iii) partial or limitingreductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography (see, e.g., McDonagh et al(2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al(2004) Clin. Cancer Res. 10:7063-7070; Hamblett, K. J., et al. “Effectof drug loading on the pharmacology, pharmacokinetics, and toxicity ofan anti-CD30 antibody-drug conjugate,” Abstract No. 624, AmericanAssociation for Cancer Research, 2004 Annual Meeting, Mar. 27-31, 2004,Proceedings of the AACR, Volume 45, March 2004; Alley, S. C., et al.“Controlling the location of drug attachment in antibody-drugconjugates,” Abstract No. 627, American Association for Cancer Research,2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume45, March 2004). In certain embodiments, a homogeneous ADC with a singleloading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

e. Certain Methods of Preparing Immunconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in US 2005-0238649 A1, which isexpressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into an antibodyby introducing one, two, three, four, or more cysteine residues (e.g.,by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic substituents on thelinker reagent or drug. In another embodiment, the sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither galactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the antibody that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Nucleophilic groups on a drug moiety include, but are not limited to:amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide groups capable of reacting toform covalent bonds with electrophilic groups on linker moieties andlinker reagents including: (i) active esters such as NHS esters, HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A; see pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

Immunoconjugates comprising an antibody and a cytotoxic agent may alsobe made using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Alternatively, a fusion protein comprising an antibody and a cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.A recombinant DNA molecule may comprise regions encoding the antibodyand cytotoxic portions of the conjugate either adjacent to one anotheror separated by a region encoding a linker peptide which does notdestroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a radionucleotide).

Exemplary Immunoconjugates—Thio-Antibody Drug Conjugates

a. Preparation of Cysteine Engineered Anti-FcRH5 Antibodies

DNA encoding an amino acid sequence variant of the cysteine engineeredanti-FcRH5 antibodies and parent anti-FcRH5 antibodies of the inventionis prepared by a variety of methods which include, but are not limitedto, isolation from a natural source (in the case of naturally occurringamino acid sequence variants), preparation by site-directed (oroligonucleotide-mediated) mutagenesis (Carter (1985) et al Nucleic AcidsRes. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; Kunkel et al(1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al (1998) J. Biol.Chem. 273:20252-20260), PCR mutagenesis (Higuchi, (1990) in PCRProtocols, pp. 177-183, Academic Press; Ito et al (1991) Gene 102:67-70;Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al(1989) Nuc. Acids Res. 17:723-733), and cassette mutagenesis (Wells etal (1985) Gene 34:315-323) of an earlier prepared DNA encoding thepolypeptide. Mutagenesis protocols, kits, and reagents are commerciallyavailable, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit(Stratagene, La Jolla, Calif.). Single mutations are also generated byoligonucleotide directed mutagenesis using double stranded plasmid DNAas template by PCR based mutagenesis (Sambrook and Russel, (2001)Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983)Methods Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl.Acids Res. 10:6487-6500). Variants of recombinant antibodies may beconstructed also by restriction fragment manipulation or by overlapextension PCR with synthetic oligonucleotides. Mutagenic primers encodethe cysteine codon replacement(s). Standard mutagenesis techniques canbe employed to generate DNA encoding such mutant cysteine engineeredantibodies (Sambrook et al Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel et al Current Protocols in Molecular Biology, Greene Publishingand Wiley-Interscience, New York, N.Y., 1993).

Phage display technology (McCafferty et al (1990) Nature 348:552-553)can be used to produce anti-FcRH5 human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell (Johnson et al (1993) Current Opinion inStructural Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628;Marks et al (1991) J. Mol. Biol. 222:581-597; Griffith et al (1993) EMBOJ. 12:725-734; U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,573,905; U.S.Pat. No. 5,567,610; U.S. Pat. No. 5,229,275).

Anti-FcRH5 antibodies may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. The appropriate amino acid sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques (Stewart et al., Solid-Phase Peptide Synthesis, (1969) W.H.Freeman Co., San Francisco, Calif.; Merrifield, (1963) J. Am. Chem.Soc., 85:2149-2154). In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated solid phase synthesis maybe accomplished, for instance, employing t-BOC or Fmoc protected aminoacids and using an Applied Biosystems Peptide Synthesizer (Foster City,Calif.) using manufacturer's instructions. Various portions of theanti-FcRH5 antibody or FcRH5 polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe desired anti-FcRH5 antibody or FcRH5 polypeptide.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (Morimoto et al (1992) Journal ofBiochemical and Biophysical Methods 24:107-117; and Brennan et al (1985)Science, 229:81), or produced directly by recombinant host cells. Fab,Fv and ScFv anti-FcRH5 antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of these fragments. Antibody fragments can be isolated from theantibody phage libraries discussed herein. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (Carter et al (1992) Bio/Technology10:163-167), or isolated directly from recombinant host cell culture.The anti-FcRH5 antibody may be a (scFv) single chain Fv fragment (WO93/16185; U.S. Pat. No. 5,571,894; U.S. Pat. No. 5,587,458). Theanti-FcRH5 antibody fragment may also be a “linear antibody” (U.S. Pat.No. 5,641,870). Such linear antibody fragments may be monospecific orbispecific.

The description below relates primarily to production of anti-FcRH5antibodies by culturing cells transformed or transfected with a vectorcontaining anti-FcRH5 antibody-encoding nucleic acid. DNA encodinganti-FcRH5 antibodies may be obtained from a cDNA library prepared fromtissue believed to possess the anti-FcRH5 antibody mRNA and to expressit at a detectable level. Accordingly, human anti-FcRH5 antibody orFcRH5 polypeptide DNA can be conveniently obtained from a cDNA libraryprepared from human tissue. The anti-FcRH5 antibody-encoding gene mayalso be obtained from a genomic library or by known synthetic procedures(e.g., automated nucleic acid synthesis).

The design, selection, and preparation methods of the invention enablecysteine engineered anti-FcRH5 antibodies which are reactive withelectrophilic functionality. These methods further enable antibodyconjugate compounds such as antibody-drug conjugate (ADC) compounds withdrug molecules at designated, designed, selective sites. Reactivecysteine residues on an antibody surface allow specifically conjugatinga drug moiety through a thiol reactive group such as maleimide orhaloacetyl. The nucleophilic reactivity of the thiol functionality of aCys residue to a maleimide group is about 1000 times higher compared toany other amino acid functionality in a protein, such as amino group oflysine residues or the N-terminal amino group. Thiol specificfunctionality in iodoacetyl and maleimide reagents may react with aminegroups, but higher pH (>9.0) and longer reaction times are required(Garman, 1997, Non-Radioactive Labelling: A Practical Approach, AcademicPress, London). The amount of free thiol in a protein may be estimatedby the standard Ellman's assay. Immunoglobulin M is an example of adisulfide-linked pentamer, while immunoglobulin G is an example of aprotein with internal disulfide bridges bonding the subunits together.In proteins such as this, reduction of the disulfide bonds with areagent such as dithiothreitol (DTT) or selenol (Singh et al (2002)Anal. Biochem. 304:147-156) is required to generate the reactive freethiol. This approach may result in loss of antibody tertiary structureand antigen binding specificity.

The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) Assayallows for detection of reactive cysteine groups in antibodies in anELISA phage format thereby assisting in the design of cysteineengineered antibodies (Junutula, J. R. et al. (2008) J Immunol Methods332:41-52; WO 2006/034488; US 2007/0092940). The cysteine engineeredantibody is coated on well surfaces, followed by incubation with phageparticles, addition of HRP labeled secondary antibody, and absorbancedetection. Mutant proteins displayed on phage may be screened in arapid, robust, and high-throughput manner. Libraries of cysteineengineered antibodies can be produced and subjected to binding selectionusing the same approach to identify appropriately reactive sites of freeCys incorporation from random protein-phage libraries of antibodies orother proteins. This technique includes reacting cysteine mutantproteins displayed on phage with an affinity reagent or reporter groupwhich is also thiol-reactive.

PHESELECTOR assay allows screening of reactive thiol groups inantibodies. Identification of the A121C variant by this method isexemplary. The entire Fab molecule may be effectively searched toidentify more ThioFab variants with reactive thiol groups. A parameter,fractional surface accessibility, was employed to identify andquantitate the accessibility of solvent to the amino acid residues in apolypeptide. The surface accessibility can be expressed as the surfacearea (Å²) that can be contacted by a solvent molecule, e.g. water. Theoccupied space of water is approximated as a 1.4 Å radius sphere.Software is freely available or licensable (Secretary to CCP4, DaresburyLaboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825,or by internet world-wide web ccp4.ac.uk/dist/html/INDEX.html) as theCCP4 Suite of crystallography programs which employ algorithms tocalculate the surface accessibility of each amino acid of a protein withknown x-ray crystallography derived coordinates (“The CCP4 Suite:Programs for Protein Crystallography” (1994) Acta. Cryst. D50:760-763).Two exemplary software modules that perform surface accessibilitycalculations are “AREAIMOL” and “SURFACE”, based on the algorithms of B.Lee and F. M. Richards (1971) J.Mol.Biol. 55:379-400. AREAIMOL definesthe solvent accessible surface of a protein as the locus of the centreof a probe sphere (representing a solvent molecule) as it rolls over theVan der Waals surface of the protein. AREAIMOL calculates the solventaccessible surface area by generating surface points on an extendedsphere about each atom (at a distance from the atom centre equal to thesum of the atom and probe radii), and eliminating those that lie withinequivalent spheres associated with neighboring atoms. AREAIMOL finds thesolvent accessible area of atoms in a PDB coordinate file, andsummarizes the accessible area by residue, by chain and for the wholemolecule. Accessible areas (or area differences) for individual atomscan be written to a pseudo-PDB output file. AREAIMOL assumes a singleradius for each element, and only recognizes a limited number ofdifferent elements.

AREAIMOL and SURFACE report absolute accessibilities, i.e. the number ofsquare Angstroms (Å). Fractional surface accessibility is calculated byreference to a standard state relevant for an amino acid within apolypeptide. The reference state is tripeptide Gly-X-Gly, where X is theamino acid of interest, and the reference state should be an ‘extended’conformation, i.e. like those in beta-strands. The extended conformationmaximizes the accessibility of X. A calculated accessible area isdivided by the accessible area in a Gly-X-Gly tripeptide reference stateand reports the quotient, which is the fractional accessibility. Percentaccessibility is fractional accessibility multiplied by 100. Anotherexemplary algorithm for calculating surface accessibility is based onthe SOLV module of the program xsae (Broger, C., F. Hoffman-LaRoche,Basel) which calculates fractional accessibility of an amino acidresidue to a water sphere based on the X-ray coordinates of thepolypeptide. The fractional surface accessibility for every amino acidin an antibody may be calculated using available crystal structureinformation (Eigenbrot et al. (1993) J Mol. Biol. 229:969-995).

DNA encoding the cysteine engineered antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, orother mammalian host cells, such as myeloma cells (U.S. Pat. No.5,807,715; US 2005/0048572; US 2004/0229310) that do not otherwiseproduce the antibody protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells.

After design and selection, cysteine engineered antibodies, e.g.ThioFabs, with the engineered, highly reactive unpaired Cys residues,“free cysteine amino acids”, may be produced by: (i) expression in abacterial, e.g. E. coli, system (Skerra et al (1993) Curr. Opinion inImmunol. 5:256-262; Pliickthun (1992) Immunol. Revs. 130:151-188) or amammalian cell culture system (WO 01/00245), e.g. Chinese Hamster Ovarycells (CHO); and (ii) purification using common protein purificationtechniques (Lowman et al (1991) J. Biol. Chem. 266(17): 10982-10988).

The engineered Cys thiol groups react with electrophilic linker reagentsand drug-linker intermediates to form cysteine engineered antibody drugconjugates and other labelled cysteine engineered antibodies. Cysresidues of cysteine engineered antibodies, and present in the parentantibodies, which are paired and form interchain and intrachaindisulfide bonds do not have any reactive thiol groups (unless treatedwith a reducing agent) and do not react with electrophilic linkerreagents or drug-linker intermediates. The newly engineered Cys residue,can remain unpaired, and able to react with, i.e. conjugate to, anelectrophilic linker reagent or drug-linker intermediate, such as adrug-maleimide. Exemplary drug-linker intermediates include: MC-MMAE,MC-MMAF, MC-vc-PAB-MMAE, and MC-vc-PAB-MMAF. The structure positions ofthe engineered Cys residues of the heavy and light chains are numberedaccording to a sequential numbering system. This sequential numberingsystem is correlated to the Kabat numbering system (Kabat et al., (1991)Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md.) starting at theN-terminus, differs from the Kabat numbering scheme (bottom row) byinsertions noted by a,b,c. Using the Kabat numbering system, the actuallinear amino acid sequence may contain fewer or additional amino acidscorresponding to a shortening of, or insertion into, a FR or CDR of thevariable domain. The cysteine engineered heavy chain variant sites areidentified by the sequential numbering and Kabat numbering schemes.

In one embodiment, the cysteine engineered anti-FcRH5 antibody isprepared by a process comprising:

(a) replacing one or more amino acid residues of a parent anti-FcRH5antibody by cysteine; and

(b) determining the thiol reactivity of the cysteine engineeredanti-FcRH5 antibody by reacting the cysteine engineered antibody with athiol-reactive reagent.

The cysteine engineered antibody may be more reactive than the parentantibody with the thiol-reactive reagent.

The free cysteine amino acid residues may be located in the heavy orlight chains, or in the constant or variable domains. Antibodyfragments, e.g. Fab, may also be engineered with one or more cysteineamino acids replacing amino acids of the antibody fragment, to formcysteine engineered antibody fragments.

Another embodiment of the invention provides a method of preparing(making) a cysteine engineered anti-FcRH5 antibody, comprising:

(a) introducing one or more cysteine amino acids into a parentanti-FcRH5 antibody in order to generate the cysteine engineeredanti-FcRH5 antibody; and

(b) determining the thiol reactivity of the cysteine engineered antibodywith a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive than theparent antibody with the thiol-reactive reagent.

Step (a) of the method of preparing a cysteine engineered antibody maycomprise:

-   -   (i) mutagenizing a nucleic acid sequence encoding the cysteine        engineered antibody;    -   (ii) expressing the cysteine engineered antibody; and    -   (iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of preparing a cysteine engineered antibody maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of preparing a cysteine engineered antibody mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

Another embodiment of the invention is a method of screening cysteineengineered antibodies with highly reactive, unpaired cysteine aminoacids for thiol reactivity comprising:

(a) introducing one or more cysteine amino acids into a parent antibodyin order to generate a cysteine engineered antibody;

(b) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(c) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media; and

(d) determining the thiol reactivity of the cysteine engineered antibodywith the thiol-reactive reagent. Step (a) of the method of screeningcysteine engineered antibodies may comprise:

(i) mutagenizing a nucleic acid sequence encoding the cysteineengineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of screening cysteine engineered antibodies maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of screening cysteine engineered antibodies mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

b. Cysteine Engineering of Anti-FcRH5 IgG Variants

Cysteine was introduced at the heavy chain 118 (EU numbering)(equivalent to heavy chain position 118, sequential numbering) (as shownin FIG. 13) site into the full-length, chimeric parent monoclonalanti-FcRH5 antibodies or at the light chain 205 (Kabat numbering) (asshown in FIG. 14) site into the full-length, chimeric parentalmonoclonal anti-FcRH5 antibodies by the cysteine engineering methodsdescribed herein.

Cysteine engineered antibodies with cysteine at heavy chain 118 (EUnumbering) generated were: (a) thio-hu13G9-HC-A118C with heavy chainsequence (SEQ ID NO: 60) and light chain sequence (SEQ ID NO: 59), FIG.13.

Cysteine engineered antibodies with cysteine at light chain 205 (Kabatnumbering) generated were: (a) thio-hu13G9-LC-V205C with heavy chainsequence (SEQ ID NO: 62) and light chain sequence (SEQ ID NO: 61), FIG.14.

c. Labelled Cysteine Engineered Anti-FcRH5 Antibodies

Cysteine engineered anti-FcRH5 antibodies may be site-specifically andefficiently coupled with a thiol-reactive reagent. The thiol-reactivereagent may be a multifunctional linker reagent, a capture, i.e.affinity, label reagent (e.g. a biotin-linker reagent), a detectionlabel (e.g. a fluorophore reagent), a solid phase immobilization reagent(e.g. SEPHAROSE™, polystyrene, or glass), or a drug-linker intermediate.One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). Inan exemplary embodiment, reaction of a ThioFab with a biotin-linkerreagent provides a biotinylated ThioFab by which the presence andreactivity of the engineered cysteine residue may be detected andmeasured. Reaction of a ThioFab with a multifunctional linker reagentprovides a ThioFab with a functionalized linker which may be furtherreacted with a drug moiety reagent or other label. Reaction of a ThioFabwith a drug-linker intermediate provides a ThioFab drug conjugate.

The exemplary methods described here may be applied generally to theidentification and production of antibodies, and more generally, toother proteins through application of the design and screening stepsdescribed herein.

Such an approach may be applied to the conjugation of otherthiol-reactive reagents in which the reactive group is, for example, amaleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive conjugation partner (Haugland, 2003, Molecular ProbesHandbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in BioconjugateTechniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). Thethiol-reactive reagent may be a drug moiety, a fluorophore such as afluorescent dye like fluorescein or rhodamine, a chelating agent for animaging or radiotherapeutic metal, a peptidyl or non-peptidyl label ordetection tag, or a clearance-modifying agent such as various isomers ofpolyethylene glycol, a peptide that binds to a third component, oranother carbohydrate or lipophilic agent.

d. Uses of Cysteine Engineered Anti-FcRH5 Antibodies

Cysteine engineered anti-FcRH5 antibodies, and conjugates thereof mayfind use as therapeutic and/or diagnostic agents. The present inventionfurther provides methods of preventing, managing, treating orameliorating one or more symptoms associated with a B-cell relateddisorder. In particular, the present invention provides methods ofpreventing, managing, treating, or ameliorating one or more symptomsassociated with a cell proliferative disorder, such as cancer, e.g.,lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsedaggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocyticlymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocyticleukemia (ALL), and mantle cell lymphoma. The present invention stillfurther provides methods for diagnosing a FcRH5 related disorder orpredisposition to developing such a disorder, as well as methods foridentifying antibodies, and antigen-binding fragments of antibodies,that preferentially bind B cell-associated FcRH5 polypeptides.

Another embodiment of the present invention is directed to the use of acysteine engineered anti-FcRH5 antibody for the preparation of amedicament useful in the treatment of a condition which is responsive toa B cell related disorder.

E. Cysteine Engineered Antibody Drug Conjugates (Thio-Antibody DrugConjugates (TDCs))

Another aspect of the invention is an antibody-drug conjugate compoundcomprising a cysteine engineered anti-FcRH5 antibody (Ab), and anauristatin drug moiety (D) wherein the cysteine engineered antibody isattached through one or more free cysteine amino acids by a linkermoiety (L) to D; the compound having Formula I:Ab-(L-D)_(p)  Iwhere p is 1, 2, 3, or 4; and wherein the cysteine engineered antibodyis prepared by a process comprising replacing one or more amino acidresidues of a parent anti-FcRH5 antibody by one or more free cysteineamino acids.

Another aspect of the invention is a composition comprising a mixture ofantibody-drug compounds of Formula I where the average drug loading perantibody is about 2 to about 5, or about 3 to about 4.

Potential advantages of cysteine engineered anti-FcRH5 antibody drugconjugates include improved safety (larger therapeutic index), improvedPK parameters, the antibody inter-chain disulfide bonds are retainedwhich may stabilize the conjugate and retain its active bindingconformation, the sites of drug conjugation are defined, and thepreparation of cysteine engineered antibody drug conjugates fromconjugation of cysteine engineered antibodies to drug-linker reagentsresults in a more homogeneous product.

Linkers

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments, a linker is specified as L. A“Linker” (L) is a bifunctional or multifunctional moiety which can beused to link one or more Drug moieties (D) and an antibody unit (Ab) toform antibody-drug conjugates (ADC) of Formula I. Antibody-drugconjugates (ADC) can be conveniently prepared using a Linker havingreactive functionality for binding to the Drug and to the Antibody. Acysteine thiol of a cysteine engineered antibody (Ab) can form a bondwith an electrophilic functional group of a linker reagent, a drugmoiety or drug-linker intermediate.

In one aspect, a Linker has a reactive site which has an electrophilicgroup that is reactive to a nucleophilic cysteine present on anantibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a Linker and forms a covalent bond to a Linker.Useful electrophilic groups include, but are not limited to, maleimideand haloacetamide groups.

Linkers include a divalent radical such as an alkyldiyl, an arylene, aheteroarylene, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

Cysteine engineered antibodies react with linker reagents or drug-linkerintermediates, with electrophilic functional groups such as maleimide orα-halo carbonyl, according to the conjugation method at page 766 ofKlussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe” or “af”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”), ethyleneoxy —CH₂CH₂O— as one ormore repeating units (“EO” or “PEO”). Additional linker components areknown in the art and some are described herein.

In one embodiment, linker L of an ADC has the formula:-A_(a)-W_(w)—Y_(y)-

wherein:

A- is a Stretcher unit covalently attached to a cysteine thiol of theantibody (Ab);

a is 0 or 1;

each —W— is independently an Amino Acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a Spacer unit covalently attached to the drug moiety; and

y is 0, 1 or 2.

Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking anantibody unit to an amino acid unit (—W—). In this regard an antibody(Ab) has a functional group that can form a bond with a functional groupof a Stretcher. Useful functional groups that can be present on anantibody, either naturally or via chemical manipulation include, but arenot limited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomerichydroxyl group of a carbohydrate, and carboxyl. In one aspect, theantibody functional groups are sulfhydryl or amino. Sulfhydryl groupscan be generated by reduction of an intramolecular disulfide bond of anantibody. Alternatively, sulfhydryl groups can be generated by reactionof an amino group of a lysine moiety of an antibody using2-iminothiolane (Traut's reagent) or another sulfhydryl generatingreagent. In one embodiment, an antibody (Ab) has a free cysteine thiolgroup that can form a bond with an electrophilic functional group of aStretcher Unit. Exemplary stretcher units in Formula I conjugates aredepicted by Formulas II and III, wherein Ab-, —W—, —Y—, -D, w and y areas defined above, and R¹⁷ is a divalent radical selected from (CH₂)_(r),C₃-C₈ carbocyclyl, O—(CH₂)_(r), arylene, (CH₂)_(r)-arylene,-arylene-(CH₂)_(r)—, (CH₂)_(r)—(C₃-C₈ carbocyclyl), (C₃-C₈carbocyclyl)-(CH₂)_(r), C₃-C₈ heterocyclyl, (CH₂)—(C₃-C₈ heterocyclyl),—(C₃-C₈ heterocyclyl)-(CH₂)_(r)—, —(CH₂)_(r)C(O)NR^(b)(CH₂)_(r)—,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—, and—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂)—; where R^(b) is H, C₁-C₆ alkyl, phenyl,or benzyl; and r is independently an integer ranging from 1-10.

Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbonatoms derived by the removal of two hydrogen atoms from the aromaticring system. Typical arylene groups include, but are not limited to,radicals derived from benzene, substituted benzene, naphthalene,anthracene, biphenyl, and the like.

Heterocyclyl groups include a ring system in which one or more ringatoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system. Heterocycles are described in Paquette, Leo A.; “Principles ofModern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,3-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromenyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

Carbocyclyl groups include a saturated or unsaturated ring having 3 to 7carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples ofmonocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

It is to be understood from all the exemplary embodiments of Formula IADC such as II-VI, that even where not denoted expressly, from 1 to 4drug moieties are linked to an antibody (p=1-4), depending on the numberof engineered cysteine residues.

An illustrative Formula II Stretcher unit is derived frommaleimido-caproyl (MC) wherein R¹⁷ is —(CH₂)₅—:

An illustrative Stretcher unit of Formula II, and is derived frommaleimido-propanoyl (MP) wherein R¹⁷ is —(CH₂)₂—:

Another illustrative Stretcher unit of Formula II wherein R¹⁷ is—(CH₂CH₂O)_(r)—CH₂— and r is 2:

Another illustrative Stretcher unit of Formula II wherein R¹⁷ is—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— where R^(b) is H and each r is 2:

An illustrative Stretcher unit of Formula III wherein R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the cysteineengineered anti-FcRH5 antibody via a disulfide bond between theengineered cysteine sulfur atom of the antibody and a sulfur atom of theStretcher unit. A representative Stretcher unit of this embodiment isdepicted by Formula IV, wherein R¹⁷, Ab-, —W—, —Y—, -D, w and y are asdefined above.

In yet another embodiment, the reactive group of the Stretcher containsa thiol-reactive functional group that can form a bond with a freecysteine thiol of an antibody. Examples of thiol-reaction functionalgroups include, but are not limited to, maleimide, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted byFormulas Va and Vb, wherein —R¹⁷—, Ab-, —W—, —Y—, -D, w and y are asdefined above;

In another embodiment, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) TetrahedronLetters 43:1987-1990). Dendritic linkers can increase the molar ratio ofdrug to antibody, i.e. loading, which is related to the potency of theADC. Thus, where a cysteine engineered antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Amino Acid Unit

The linker may comprise amino acid residues. The Amino Acid unit(—W_(w)—), when present, links the antibody (Ab) to the drug moiety (D)of the cysteine engineered antibody-drug conjugate (ADC) of theinvention.

—W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit Amino acid residues which comprisethe Amino Acid unit include those occurring naturally, as well as minoramino acids and non-naturally occurring amino acid analogs, such ascitrulline. Each —W— unit independently has the formula denoted below inthe square brackets, and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

When R¹⁹ is other than hydrogen, the carbon atom to which R¹⁹ isattached is chiral. Each carbon atom to which R¹⁹ is attached isindependently in the (S) or (R) configuration, or a racemic mixtureAmino acid units may thus be enantiomerically pure, racemic, ordiastereomeric.

Exemplary —W_(w)— Amino Acid units include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly) Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline.

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug moiety (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D). Amino acid linker components can be designed and optimized intheir selectivity for enzymatic cleavage by a particular enzymes, forexample, a tumor-associated protease, cathepsin B, C and D, or a plasminprotease.

Spacer Unit

The Spacer unit (—Y_(r)—), when present (y=1 or 2), links an Amino Acidunit (—W—) to the drug moiety (D) when an Amino Acid unit is present(w=1-12). Alternately, the Spacer unit links the Stretcher unit to theDrug moiety when the Amino Acid unit is absent. The Spacer unit alsolinks the drug moiety to the antibody unit when both the Amino Acid unitand Stretcher unit are absent (w, y=0). Spacer units are of two generaltypes: self-immolative and non self-immolative. A non self-immolativeSpacer unit is one in which part or all of the Spacer unit remains boundto the Drug moiety after cleavage, particularly enzymatic, of an AminoAcid unit from the antibody-drug conjugate or the Drug moiety-linker.When an ADC containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved fromAb-A_(a)-W_(w)—. In one embodiment, an independent hydrolysis reactiontakes place within the target cell, cleaving the glycine-Drug moietybond and liberating the Drug.

In another embodiment, —Y_(r)— is a p-aminobenzylcarbamoyl (PAB) unitwhose phenylene portion is substituted with Q_(m) wherein Q is —C₁-C₈alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; and m is an integerranging from 0-4.

Exemplary embodiments of a non self-immolative Spacer unit (—Y—) are:-Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one embodiment, a Drug moiety-linker or an ADC is provided in whichthe Spacer unit is absent (y=0), or a pharmaceutically acceptable saltor solvate thereof.

Alternatively, an ADC containing a self-immolative Spacer unit canrelease -D. In one embodiment, —Y— is a PAB group that is linked to—W_(w)— via the amino nitrogen atom of the PAB group, and connecteddirectly to -D via a carbonate, carbamate or ether group, where the ADChas the exemplary structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p ranges from 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237), heterocyclic PAB analogs (US2005/0256030), beta-glucuronide (WO 2007/011968), and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ringsystems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and2-aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem.55:5867). Elimination of amine-containing drugs that are substituted atglycine (Kingsbury et al (1984) J. Med. Chem. 27:1447) are also examplesof self-immolative spacer useful in ADCs.

Exemplary Spacer units (—Y_(y)—) are represented by Formulas X-XII:

Dendritic Linkers

In another embodiment, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the ADC. Thus, where a cysteine engineeredantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker. Exemplaryembodiments of branched, dendritic linkers include2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenoldendrimer units (WO 2004/01993; Szalai et al (2003) J. Amer. Chem. Soc.125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126:1726-1731;Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499).

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS), which can be used to incorporate andrelease multiple drugs, having the structure:

comprising a 2-(4-aminobenzylidene)propane-1,3-diol dendrimer unit (WO2004/043493; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494),wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges ranging from1 to 4.

Exemplary embodiments of the Formula I antibody-drug conjugate compoundsinclude XIIIa (MC), XIIIb (val-cit), XIIIc (MC-val-cit), and XIIId(MC-val-cit-PAB):

Other exemplary embodiments of the Formula Ia antibody-drug conjugatecompounds include XIVa-e:

and R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In another embodiment, a Linker has a reactive functional group whichhas a nucleophilic group that is reactive to an electrophilic grouppresent on an antibody. Useful electrophilic groups on an antibodyinclude, but are not limited to, aldehyde and ketone carbonyl groups.The heteroatom of a nucleophilic group of a Linker can react with anelectrophilic group on an antibody and form a covalent bond to anantibody unit. Useful nucleophilic groups on a Linker include, but arenot limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on anantibody provides a convenient site for attachment to a Linker.

Typically, peptide-type Linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press) which is well known in the field ofpeptide chemistry. Linker intermediates may be assembled with anycombination or sequence of reactions including Spacer, Stretcher, andAmino Acid units. The Spacer, Stretcher, and Amino Acid units may employreactive functional groups which are electrophilic, nucleophilic, orfree radical in nature. Reactive functional groups include, but are notlimited to carboxyls, hydroxyls, para-nitrophenylcarbonate,isothiocyanate, and leaving groups, such as O-mesyl, O-tosyl, —Cl, —Br,—I; or maleimide.

For example, a charged substituent such as sulfonate (—SO₃ ⁻) orammonium, may increase water solubility of the reagent and facilitatethe coupling reaction of the linker reagent with the antibody or thedrug moiety, or facilitate the coupling reaction of Ab-L(antibody-linker intermediate) with D, or D-L (drug-linker intermediate)with Ab, depending on the synthetic route employed to prepare the ADC.

Linker Reagents

Conjugates of the antibody and auristatin may be made using a variety ofbifunctional linker reagents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The antibody drug conjugates may also be prepared with linker reagents:BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, 1,8-bis-maleimidodiethyleneglycol(BM(PEO)₂), and 1,11-bis-maleimidotriethyleneglycol (BM(PEO)₃), whichare commercially available from Pierce Biotechnology, Inc.,ThermoScientific, Rockford, Ill., and other reagent suppliers.Bis-maleimide reagents allow the attachment of the thiol group of acysteine engineered antibody to a thiol-containing drug moiety, label,or linker intermediate, in a sequential or concurrent fashion. Otherfunctional groups besides maleimide, which are reactive with a thiolgroup of a cysteine engineered antibody, drug moiety, label, or linkerintermediate include iodoacetamide, bromoacetamide, vinyl pyridine,disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

Useful linker reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem.60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; U.S.Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO03/026577; WO 03/043583; and WO 04/032828.

Stretchers of formula (Ma) can be introduced into a Linker by reactingthe following linker reagents with the N-terminus of an Amino Acid unit:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of can be introduced into a Linker by reacting thefollowing bifunctional reagents with the N-terminus of an Amino Acidunit:

where X is Br or I.

Stretcher units of formula can also be introduced into a Linker byreacting the following bifunctional reagents with the N-terminus of anAmino Acid unit:

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide Stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative Spacer has the structure:

An exemplary phe-lys(Mtr, mono-4-methoxytrityl) dipeptide linker reagenthaving a maleimide Stretcher unit and a PAB self-immolative Spacer unitcan be prepared according to Dubowchik, et al. (1997) TetrahedronLetters, 38:5257-60, and has the structure:

Preparation of Cysteine Engineered Anti-FcRH5 Antibody-Drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a cysteine group of acysteine engineered antibody with a linker reagent, to formantibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a linker reagent, to formdrug-linker intermediate D-L, via a covalent bond, followed by reactionwith a cysteine group of a cysteine engineered antibody. Conjugationmethods (1) and (2) may be employed with a variety of cysteineengineered antibodies, drug moieties, and linkers to prepare theantibody-drug conjugates of Formula I.

Antibody cysteine thiol groups are nucleophilic and capable of reactingto form covalent bonds with electrophilic groups on linker reagents anddrug-linker intermediates including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides, such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups; and (iv) disulfides, including pyridyldisulfides, via sulfide exchange. Nucleophilic groups on a drug moietyinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

Cysteine engineered antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.), followed byreoxidation to reform interchain and intrachain disulfide bonds (Example5). For example, full length, cysteine engineered monoclonal antibodies(ThioMabs) expressed in CHO cells are reduced with about a 50 fold molarexcess of TCEP for 3 hrs at 37° C. to reduce disulfide bonds in cysteineadducts which may form between the newly introduced cysteine residuesand the cysteine present in the culture media. The reduced ThioMab isdiluted and loaded onto HiTrap S column in 10 mM sodium acetate, pH 5,and eluted with PBS containing 0.3M sodium chloride. Disulfide bondswere reestablished between cysteine residues present in the parent Mabwith dilute (200 nM) aqueous copper sulfate (CuSO₄) at room temperature,overnight. Alternatively, dehydroascorbic acid (DHAA) is an effectiveoxidant to reestablish the intrachain disulfide groups of the cysteineengineered antibody after reductive cleavage of the cysteine adducts.Other oxidants, i.e. oxidizing agents, and oxidizing conditions, whichare known in the art may be used. Ambient air oxidation is alsoeffective. This mild, partial reoxidation step forms intrachaindisulfides efficiently with high fidelity and preserves the thiol groupsof the newly introduced cysteine residues. An approximate 10 fold excessof drug-linker intermediate, e.g. MC-vc-PAB-MMAE, was added, mixed, andlet stand for about an hour at room temperature to effect conjugationand form the anti-FcRH5 antibody-drug conjugate. The conjugation mixturewas gel filtered and loaded and eluted through a HiTrap S column toremove excess drug-linker intermediate and other impurities.

A general process to prepare a cysteine engineered antibody expressedfrom cell culture for conjugation is as follows. When the cell culturemedia contains cysteine, disulfide adducts can form between the newlyintroduced cysteine amino acid and cysteine from media. These cysteineadducts, depicted as a circle in the exemplary ThioMab (left) in FIG.23, must be reduced to generate cysteine engineered antibodies reactivefor conjugation. Cysteine adducts, presumably along with variousinterchain disulfide bonds, are reductively cleaved to give a reducedform of the antibody with reducing agents such as TCEP. The interchaindisulfide bonds between paired cysteine residues are reformed underpartial oxidation conditions with copper sulfate, DHAA, or exposure toambient oxygen. The newly introduced, engineered, and unpaired cysteineresidues remain available for reaction with linker reagents ordrug-linker intermediates to form the antibody conjugates of theinvention. The ThioMabs expressed in mammalian cell lines result inexternally conjugated Cys adduct to an engineered Cys through —S—S— bondformation. Hence the purified ThioMabs are treated with the reductionand reoxidation procedures to produce reactive ThioMabs. These ThioMabsare used to conjugate with maleimide containing cytotoxic drugs,fluorophores, and other labels.

10. Immunoliposomes

The anti-FcRH5 antibodies disclosed herein may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

B. Certain Methods of Making Antibodies

1. Screening for Anti-FcRH5 Antibodies With the Desired Properties

Techniques for generating antibodies that bind to FcRH5 polypeptideshave been described above. One may further select antibodies withcertain biological characteristics, as desired.

The growth inhibitory effects of an anti-FcRH5 antibody of the inventionmay be assessed by methods known in the art, e.g., using cells whichexpress a FcRH5 polypeptide either endogenously or followingtransfection with the FcRH5 gene. For example, appropriate tumor celllines and FcRH5-transfected cells may be treated with an anti-FcRH5monoclonal antibody of the invention at various concentrations for a fewdays (e.g., 2-7) days and stained with crystal violet or MTT or analyzedby some other colorimetric assay. Another method of measuringproliferation would be by comparing ³H-thymidine uptake by the cellstreated in the presence or absence an anti-FcRH5 antibody of theinvention. After treatment, the cells are harvested and the amount ofradioactivity incorporated into the DNA quantitated in a scintillationcounter. Appropriate positive controls include treatment of a selectedcell line with a growth inhibitory antibody known to inhibit growth ofthat cell line. Growth inhibition of tumor cells in vivo can bedetermined in various ways known in the art. The tumor cell may be onethat overexpresses a FcRH5 polypeptide. The anti-FcRH5 antibody willinhibit cell proliferation of a FcRH5-expressing tumor cell in vitro orin vivo by about 25-100% compared to the untreated tumor cell, morepreferably, by about 30-100%, and even more preferably by about 50-100%or 70-100%, in one embodiment, at an antibody concentration of about 0.5to 30 μg/ml. Growth inhibition can be measured at an antibodyconcentration of about 0.5 to 30 μg/ml or about 0.5 nM to 200 nM in cellculture, where the growth inhibition is determined 1-10 days afterexposure of the tumor cells to the antibody. The antibody is growthinhibitory in vivo if administration of the anti-FcRH5 antibody at about1 μg/kg to about 100 mg/kg body weight results in reduction in tumorsize or reduction of tumor cell proliferation within about 5 days to 3months from the first administration of the antibody, preferably withinabout 5 to 30 days.

To select for an anti-FcRH5 antibody which induces cell death, loss ofmembrane integrity as indicated by, e.g., propidium iodide (PI), trypanblue or 7AAD uptake may be assessed relative to control. A PI uptakeassay can be performed in the absence of complement and immune effectorcells. FcRH5 polypeptide-expressing tumor cells are incubated withmedium alone or medium containing the appropriate anti-FcRH5 antibody(e.g, at about 10 μg/ml). The cells are incubated for a 3 day timeperiod. Following each treatment, cells are washed and aliquoted into 35mm strainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT®CellQuest software (Becton Dickinson). Those anti-FcRH5 antibodies thatinduce statistically significant levels of cell death as determined byPI uptake may be selected as cell death-inducing anti-FcRH5 antibodies.

To screen for antibodies which bind to an epitope on a FcRH5 polypeptidebound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Thisassay can be used to determine if a test antibody binds the same site orepitope as a known anti-FcRH5 antibody. Alternatively, or additionally,epitope mapping can be performed by methods known in the art. Forexample, the antibody sequence can be mutagenized such as by alaninescanning, to identify contact residues. The mutant antibody is initiallytested for binding with polyclonal antibody to ensure proper folding. Ina different method, peptides corresponding to different regions of aFcRH5 polypeptide can be used in competition assays with the testantibodies or with a test antibody and an antibody with a characterizedor known epitope.

2. Certain Library Screening Methods

Anti-FcRH5 antibodies of the invention can be made by usingcombinatorial libraries to screen for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are described generally in Hoogenboom et al. (2001) inMethods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,Totowa, N.J.), and in certain embodiments, in Lee et al. (2004) J. Mol.Biol. 340:1073-1093.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the anti-FcRH5 antibodies of the inventioncan be obtained by designing a suitable antigen screening procedure toselect for the phage clone of interest followed by construction of afull length anti-FcRH5 antibody clone using the Fv sequences from thephage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-FcRH5 clones is desired, the subject is immunized withFcRH5 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-FcRH5 clones isobtained by generating an anti-FcRH5 antibody response in transgenicmice carrying a functional human immunoglobulin gene array (and lackinga functional endogenous antibody production system) such that FcRH5immunization gives rise to B cells producing human antibodies againstFcRH5. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-FcRH5 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing FcRH5-specific membrane bound antibody, e.g., by cellseparation using FcRH5 affinity chromatography or adsorption of cells tofluorochrome-labeled FcRH5 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which FcRH5 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Santry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, FcRH5 can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized FcRH5 underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by FcRH5 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for FcRH5.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting FcRH5, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated FcRH5, but with the biotinylated FcRH5 at a concentrationof lower molarity than the target molar affinity constant for FcRH5. Thehigh affinity-binding phages can then be captured by streptavidin-coatedparamagnetic beads. Such “equilibrium capture” allows the antibodies tobe selected according to their affinities of binding, with sensitivitythat permits isolation of mutant clones with as little as two-foldhigher affinity from a great excess of phages with lower affinity.Conditions used in washing phages bound to a solid phase can also bemanipulated to discriminate on the basis of dissociation kinetics.

Anti-FcRH5 clones may be selected based on activity. In certainembodiments, the invention provides anti-FcRH5 antibodies that bind toliving cells that naturally express FcRH5. In one embodiment, theinvention provides anti-FcRH5 antibodies that block the binding betweena FcRH5 ligand and FcRH5, but do not block the binding between a FcRH5ligand and a second protein. Fv clones corresponding to such anti-FcRH5antibodies can be selected by (1) isolating anti-FcRH5 clones from aphage library as described above, and optionally amplifying the isolatedpopulation of phage clones by growing up the population in a suitablebacterial host; (2) selecting FcRH5 and a second protein against whichblocking and non-blocking activity, respectively, is desired; (3)adsorbing the anti-FcRH5 phage clones to immobilized FcRH5; (4) using anexcess of the second protein to elute any undesired clones thatrecognize FcRH5-binding determinants which overlap or are shared withthe binding determinants of the second protein; and (5) eluting theclones which remain adsorbed following step (4). Optionally, clones withthe desired blocking/non-blocking properties can be further enriched byrepeating the selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-FcRH5 antibody derived from a hybridoma can also bemodified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g. as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNAencoding a hybridoma- or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies of the invention.

C. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)

The antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to anactive anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared asdescribed herein for delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the anti-FcRH5antibodies by techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., Nature 312:604-608 (1984).

D. Anti-FcRH5 Antibody

In addition to the anti-FcRH5 antibodies described herein, it iscontemplated that anti-FcRH5 antibody variants can be prepared.Anti-FcRH5 antibody variants can be prepared by introducing appropriatenucleotide changes into the encoding DNA, and/or by synthesis of thedesired antibody or polypeptide. Those skilled in the art willappreciate that amino acid changes may alter post-translationalprocesses of the anti-FcRH5 antibody, such as changing the number orposition of glycosylation sites or altering the membrane anchoringcharacteristics.

Variations in the anti-FcRH5 antibodies described herein, can be made,for example, using any of the techniques and guidelines for conservativeand non-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the antibody or polypeptide that results ina change in the amino acid sequence as compared with the native sequenceantibody or polypeptide. Optionally the variation is by substitution ofat least one amino acid with any other amino acid in one or more of thedomains of the anti-FcRH5 antibody. Guidance in determining which aminoacid residue may be inserted, substituted or deleted without adverselyaffecting the desired activity may be found by comparing the sequence ofthe anti-FcRH5 antibody with that of homologous known protein moleculesand minimizing the number of amino acid sequence changes made in regionsof high homology. Amino acid substitutions can be the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

Anti-FcRH5 antibody fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native antibodyor protein. Certain fragments lack amino acid residues that are notessential for a desired biological activity of the anti-FcRH5 antibody.

Anti-FcRH5 antibody fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating antibody orpolypeptide fragments by enzymatic digestion, e.g., by treating theprotein with an enzyme known to cleave proteins at sites defined byparticular amino acid residues, or by digesting the DNA with suitablerestriction enzymes and isolating the desired fragment. Yet anothersuitable technique involves isolating and amplifying a DNA fragmentencoding a desired antibody or polypeptide fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, anti-FcRH5 antibody fragments share at least one biologicaland/or immunological activity with the native anti-FcRH5 antibodydisclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 8 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 8, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 8 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of theanti-FcRH5 antibody are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the anti-FcRH5 antibody variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the anti-FcRH5 antibody also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking Conversely, cysteine bond(s) may be added to theanti-FcRH5 antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and FcRH5 polypeptide. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theanti-FcRH5 antibody are prepared by a variety of methods known in theart. These methods include, but are not limited to, isolation from anatural source (in the case of naturally occurring amino acid sequencevariants) or preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the anti-FcRH5 antibody.

E. Modifications of Anti-FcRH5 Antibodies

Covalent modifications of anti-FcRH5 antibodies are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of an anti-FcRH5 antibody with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of the anti-FcRH5 antibody.Derivatization with bifunctional agents is useful, for instance, forcrosslinking anti-FcRH5 antibody to a water-insoluble support matrix orsurface for use in the method for purifying anti-FcRH5 antibodies, andvice-versa. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the anti-FcRH5 antibodyincluded within the scope of this invention comprises altering thenative glycosylation pattern of the antibody or polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequenceanti-FcRH5 antibody (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence anti-FcRH5 antibody. In addition, thephrase includes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

Glycosylation of antibodies and other polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the anti-FcRH5 antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the original anti-FcRH5 antibody (forO-linked glycosylation sites). The anti-FcRH5 antibody amino acidsequence may optionally be altered through changes at the DNA level,particularly by mutating the DNA encoding the anti-FcRH5 antibody atpreselected bases such that codons are generated that will translateinto the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theanti-FcRH5 antibody is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the anti-FcRH5 antibody maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification of anti-FcRH5 antibody compriseslinking the antibody to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The antibodyalso may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

The anti-FcRH5 antibody of the present invention may also be modified ina way to form chimeric molecules comprising an anti-FcRH5 antibody fusedto another, heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theanti-FcRH5 antibody with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the anti-FcRH5antibody. The presence of such epitope-tagged forms of the anti-FcRH5antibody can be detected using an antibody against the tag polypeptide.Also, provision of the epitope tag enables the anti-FcRH5 antibody to bereadily purified by affinity purification using an anti-tag antibody oranother type of affinity matrix that binds to the epitope tag. Varioustag polypeptides and their respective antibodies are well known in theart. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the anti-FcRH5 antibody with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an “immunoadhesin”), such a fusion couldbe to the Fc region of an IgG molecule. The Ig fusions preferablyinclude the substitution of a soluble (transmembrane domain deleted orinactivated) form of an anti-FcRH5 antibody in place of at least onevariable region within an Ig molecule. In a particularly preferredembodiment, the immunoglobulin fusion includes the hinge, CH₂ and CH₃,or the hinge, CH₁, CH₂ and CH₃ regions of an IgG1 molecule. For theproduction of immunoglobulin fusions see also U.S. Pat. No. 5,428,130issued Jun. 27, 1995.

F. Preparation of Anti-FcRH5 Antibodies

The description below relates primarily to production of anti-FcRH5antibodies by culturing cells transformed or transfected with a vectorcontaining anti-FcRH5 antibody-encoding nucleic acid. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare anti-FcRH5 antibodies. For instance, theappropriate amino acid sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theanti-FcRH5 antibody may be chemically synthesized separately andcombined using chemical or enzymatic methods to produce the desiredanti-FcRH5 antibody.

1. Isolation of DNA Encoding Anti-FcRH5 Antibody

DNA encoding anti-FcRH5 antibody may be obtained from a cDNA libraryprepared from tissue believed to possess the anti-FcRH5 antibody mRNAand to express it at a detectable level. Accordingly, human anti-FcRH5antibody DNA can be conveniently obtained from a cDNA library preparedfrom human tissue. The anti-FcRH5 antibody-encoding gene may also beobtained from a genomic library or by known synthetic procedures (e.g.,automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding anti-FcRH5 antibody is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

Techniques for screening a cDNA library are well known in the art. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for anti-FcRH5 antibody production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation, which means introduction of DNA into the host so thatthe DNA is replicable, either as an extrachromosomal or by chromosomalintegrant, are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated, polyethylene-gycol/DMSO andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published 29 Jun. 1989. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transfections have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells.

a. Prokaryotic Host Cells

Suitable prokaryotes include but are not limited to archaebacteria andeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as E. coli. Various E. coli strains arepublicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E.coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772(ATCC 53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, Rhizobia, Vitreoscilla, Paracoccus and Streptomyces. Theseexamples are illustrative rather than limiting. Strain W3110 is oneparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell secretes minimal amounts of proteolytic enzymes. For example,strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2(Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No. 27,325) may be modified to effect a geneticmutation in the genes encoding proteins endogenous to the host, withexamples of such hosts including E. coli W3110 strain 1A2, which has thecomplete genotype tonA; E. coli W3110 strain 9E4, which has the completegenotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan^(r); E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ; E. coli W3110strain 40B4, which is strain 37D6 with a non-kanamycin resistant degPdeletion mutation; E. coli W3110 strain 33D3 having genotype W3110 ΔfhuA(ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kan^(R) (U.S. Pat.No. 5,639,635) and an E. coli strain having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Other strainsand derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B,E. coli _(λ) 1776 (ATCC 31,537) and E. coli RV308(ATCC 31,608) are alsosuitable. These examples are illustrative rather than limiting. Methodsfor constructing derivatives of any of the above-mentioned bacteriahaving defined genotypes are known in the art and described in, forexample, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli is faster and more cost efficient. For expression of antibodyfragments and polypeptides in bacteria, see, e.g., U.S. Pat. No.5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), andU.S. Pat. No. 5,840,523 (Simmons et al.) which describes translationinitiation regio (TIR) and signal sequences for optimizing expressionand secretion, these patents incorporated herein by reference. Afterexpression, the antibody is isolated from the E. coli cell paste in asoluble fraction and can be purified through, e.g., a protein A or Gcolumn depending on the isotype. Final purification can be carried outsimilar to the process for purifying antibody expressed e.g., in CHOcells.

b. Eukaryotic Host Cells

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-FcRH5antibody-encoding vectors. Saccharomyces cerevisiae is a commonly usedlower eukaryotic host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated anti-FcRH5antibody are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells, such as cell cultures of cotton,corn, potato, soybean, petunia, tomato, and tobacco. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa califormica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-FcRH5 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

3. Selection and Use of a Replicable Vector

For recombinant production of an antibody of the invention, the nucleicacid (e.g., cDNA or genomic DNA) encoding it is isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the antibody is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody). Many vectors are available. Thechoice of vector depends in part on the host cell to be used. Generally,preferred host cells are of either prokaryotic or eukaryotic (generallymammalian) origin.

The vector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The FcRH5 may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe anti-FcRH5 antibody-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion thesignal sequence may be, e.g., the yeast invertase leader, alpha factorleader (including Saccharomyces and Kluyveromyces factor leaders, thelatter described in U.S. Pat. No. 5,010,182), or acid phosphataseleader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr.1990), or the signal described in WO 90/13646 published 15 Nov. 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

a. Prokaryotic Host Cells

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. Both expression and cloning vectors containa nucleic acid sequence that enables the vector to replicate in one ormore selected host cells, as well as marking sequences which are capableof providing phenotypic selection in transformed cells. Such sequencesare well known for a variety of bacteria, yeast, and viruses. The originof replication from the plasmid pBR322, which contains genes encodingampicillin (Amp) and tetracycline (Tet) resistance and thus provideseasy means for identifying transformed cells, is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. pBR322, its derivatives, orother microbial plasmids or bacteriophage may also contain, or bemodified to contain, promoters which can be used by the microbialorganism for expression of endogenous proteins. Examples of pBR322derivatives used for expression of particular antibodies are describedin detail in Carter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include thePhoA promoter, the β-galactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776] and hybrid promoters suchas the tac [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]or the trc promoter. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding anti-FcRH5 antibody. However, other promoters that arefunctional in bacteria (such as other known bacterial or phagepromoters) are suitable as well. Their nucleotide sequences have beenpublished, thereby enabling a skilled worker operably to ligate them tocistrons encoding the target light and heavy chains (Siebenlist et al.(1980) Cell 20: 269) using linkers or adaptors to supply any requiredrestriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantibodies of the invention. Such modulation is accomplished at least inpart by simultaneously modulating translational strengths for thepolypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence, although silent changes in the nucleotide sequenceare preferred. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desiredantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Basedon the translational strength comparison, the desired individual TIRsare selected to be combined in the expression vector constructs of theinvention.

b. Eukaryotic Host Cells

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(1) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(2) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(3) Selection Gene Component

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up theanti-FcRH5 antibody-encoding nucleic acid, such as DHFR or thymidinekinase, metallothionein-I and -II, preferably primate metallothioneingenes, adenosine deaminase, ornithine decarboxylase, etc. An appropriatehost cell when wild-type DHFR is employed is the CHO cell line deficientin DHFR activity (e.g., ATCC CRL-9096), prepared and propagated asdescribed by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980).For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR.Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp 1 gene present inthe yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157(1980)]. The trp1 gene provides a selection marker for a mutant strainof yeast lacking the ability to grow in tryptophan, for example, ATCCNo. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

(4) Promoter Component

Expression and cloning vectors usually contain a promoter operablylinked to the anti-FcRH5 antibody-encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known.

Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Anti-FcRH5 antibody transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of a DNA encoding the anti-FcRH5 antibody by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, α-fetoprotein, and insulin). Typically, however, one will usean enhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature 297:17-18 (1982) on enhancing elements for activation ofeukaryotic promoters. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the anti-FcRH5 antibody coding sequence, but ispreferably located at a site 5′ from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding anti-FcRH5 antibody. One usefultranscription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of anti-FcRH5 antibody in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Culturing the Host Cells

The host cells used to produce the anti-FcRH5 antibody of this inventionmay be cultured in a variety of media.

a. Prokaryotic Host Cells

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay. In one aspect of the invention, antibodyproduction is conducted in large quantity by a fermentation process.Various large-scale fed-batch fermentation procedures are available forproduction of recombinant proteins. Large-scale fermentations have atleast 1000 liters of capacity, preferably about 1,000 to 100,000 litersof capacity. These fermentors use agitator impellers to distributeoxygen and nutrients, especially glucose (the preferred carbon/energysource). Small scale fermentation refers generally to fermentation in afermentor that is no more than approximately 100 liters in volumetriccapacity, and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

b. Eukaryotic Host Cells

Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ((DMEM), Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham et al., Meth.Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells. Any of these media may be supplementedas necessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleotides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceFcRH5 polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to FcRH5DNA and encoding a specific antibody epitope.

6. Purification of Anti-FcRH5 Antibody

Forms of anti-FcRH5 antibody may be recovered from culture medium orfrom host cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of anti-FcRH5 antibodycan be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify anti-FcRH5 antibody from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the anti-FcRH5 antibody. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular anti-FcRH5 antibody produced.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minCell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2 or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

G. Pharmaceutical Formulations

The antibody-drug conjugates (ADC) of the invention may be administeredby any route appropriate to the condition to be treated. The ADC willtypically be administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural.

For treating these cancers, in one embodiment, the antibody-drugconjugate is administered via intravenous infusion. The dosageadministered via infusion is in the range of about 1 μg/m² to about10,000 μg/m² per dose, generally one dose per week for a total of one,two, three or four doses. Alternatively, the dosage range is of about 1μg/m² to about 1000 μg/m², about 1 μg/m² to about 800 μg/m², about 1μg/m² to about 600 μg/m², about 1 μg/m² to about 400 μg/m², about 10μg/m² to about 500 μg/m², about 10 μg/m² to about 300 μg/m², about 10μg/m² to about 200 μg/m², and about 1 μg/m² to about 200 μg/m². The dosemay be administered once per day, once per week, multiple times perweek, but less than once per day, multiple times per month but less thanonce per day, multiple times per month but less than once per week, onceper month or intermittently to relieve or alleviate symptoms of thedisease. Administration may continue at any of the disclosed intervalsuntil remission of the tumor or symptoms of the lymphoma, leukemia beingtreated. Administration may continue after remission or relief ofsymptoms is achieved where such remission or relief is prolonged by suchcontinued administration.

The invention also provides a method of alleviating an autoimmunedisease, comprising administering to a patient suffering from theautoimmune disease, a therapeutically effective amount of a humanized13G9 antibody-drug conjugate of any one of the preceding embodiments. Inpreferred embodiments the antibody is administered intravenously orsubcutaneously. The antibody-drug conjugate is administeredintravenously at a dosage in the range of about 1 μg/m² to about 100mg/m² per dose and in a specific embodiment, the dosage is 1 μg/m² toabout 500 μg/m². The dose may be administered once per day, once perweek, multiple times per week, but less than once per day, multipletimes per month but less than once per day, multiple times per month butless than once per week, once per month or intermittently to relieve oralleviate symptoms of the disease. Administration may continue at any ofthe disclosed intervals until relief from or alleviation of symptoms ofthe autoimmune disease being treated. Administration may continue afterrelief from or alleviation of symptoms is achieved where suchalleviation or relief is prolong by such continued administration.

The invention also provides a method of treating a B cell disordercomprising administering to a patient suffering from a B cell disorder,such as a B cell proliferative disorder (including without limitationlymphoma and leukemia) or an autoimmune disease, a therapeuticallyeffective amount of a humanized 13G9 antibody of any one of thepreceding embodiments, which antibody is not conjugated to a cytotoxicmolecule or a detectable molecule. The anatibody will typically beadministered in a dosage range of about 1 μg/m² to about 1000 mg/m².

In one aspect, the invention further provides pharmaceuticalformulations comprising at least one anti-FcRH5 antibody of theinvention and/or at least one immunoconjugate thereof and/or at leastone anti-FcRH5 antibody-drug conjugate of the invention. In someembodiments, a pharmaceutical formulation comprises (1) an antibody ofthe invention and/or an immunoconjugate thereof, and (2) apharmaceutically acceptable carrier. In some embodiments, apharmaceutical formulation comprises (1) an antibody of the inventionand/or an immunoconjugate thereof, and optionally, (2) at least oneadditional therapeutic agent. Additional therapeutic agents include, butare not limited to, those described below. The ADC will typically beadministered parenterally, i.e. infusion, subcutaneous, intramuscular,intravenous, intradermal, intrathecal and epidural.

Therapeutic formulations comprising an anti-FcRH5 antibody or FcRH5immunoconjugate used in accordance with the present invention areprepared for storage by mixing the antibody or immunoconjugate, havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as acetate, Tris, phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides;

proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA;tonicifiers such as trehalose and sodium chloride; sugars such assucrose, mannitol, trehalose or sorbitol; surfactant such aspolysorbate; salt-forming counter-ions such as sodium; metal complexes(e.g., Zn-protein complexes); and/or non-ionic surfactants such asTWEEN®, PLURONICS® or polyethylene glycol (PEG). Pharmaceuticalformulations to be used for in vivo administration are generallysterile. This is readily accomplished by filtration through sterilefiltration membranes.

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, in addition to an anti-FcRH5 antibody, it may bedesirable to include in the one formulation, an additional antibody,e.g., a second anti-FcRH5 antibody which binds a different epitope onthe FcRH5 polypeptide, or an antibody to some other target such as agrowth factor that affects the growth of the particular cancer.Alternatively, or additionally, the composition may further comprise achemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitoryagent, anti-hormonal agent, and/or cardioprotectant. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT®(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated immunoglobulinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

An antibody may be formulated in any suitable form for delivery to atarget cell/tissue. For example, antibodies may be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J National Cancer Inst. 81(19):1484 (1989).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

H. Treatment with Anti-FcRH5 Antibodies

To determine FcRH5 expression in the cancer, various detection assaysare available. In one embodiment, FcRH5 polypeptide overexpression maybe analyzed by immunohistochemistry (IHC). Parrafin embedded tissuesections from a tumor biopsy may be subjected to the IHC assay andaccorded a FcRH5 protein staining intensity criteria as follows:

Score O— no staining is observed or membrane staining is observed inless than 10% of tumor cells.

Score 1±—a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.

Score 2+—a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.

Score 3+—a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for FcRH5 polypeptide expression may becharacterized as not overexpressing FcRH5, whereas those tumors with 2+or 3+ scores may be characterized as overexpressing FcRH5.

Alternatively, or additionally, FISH assays such as the INFORM® (sold byVentana, Ariz.) or PATHVISION® (Vysis, Ill.) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of FcRH5 overexpression in the tumor.

FcRH5 overexpression or amplification may be evaluated using an in vivodetection assay, e.g., by administering a molecule (such as an antibody)which binds the molecule to be detected and is tagged with a detectablelabel (e.g., a radioactive isotope or a fluorescent label) andexternally scanning the patient for localization of the label.

As described above, the anti-FcRH5 antibodies of the invention havevarious non-therapeutic applications. The anti-FcRH5 antibodies of thepresent invention can be useful for staging of FcRH5polypeptide-expressing cancers (e.g., in radioimaging). The antibodiesare also useful for purification or immunoprecipitation of FcRH5polypeptide from cells, for detection and quantitation of FcRH5polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill andeliminate FcRH5-expressing cells from a population of mixed cells as astep in the purification of other cells.

Currently, depending on the stage of the cancer, cancer treatmentinvolves one or a combination of the following therapies: surgery toremove the cancerous tissue, radiation therapy, and chemotherapy.Anti-FcRH5 antibody therapy may be especially desirable in elderlypatients who do not tolerate the toxicity and side effects ofchemotherapy well and in metastatic disease where radiation therapy haslimited usefulness. The tumor targeting anti-FcRH5 antibodies of theinvention are useful to alleviate FcRH5-expressing cancers upon initialdiagnosis of the disease or during relapse. For therapeuticapplications, the anti-FcRH5 antibody can be used alone, or incombination therapy with, e.g., hormones, antiangiogens, orradiolabelled compounds, or with surgery, cryotherapy, and/orradiotherapy. Anti-FcRH5 antibody treatment can be administered inconjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy. Chemotherapeuticdrugs such as TAXOTERE® (docetaxel), TAXOL® (palictaxel), estramustineand mitoxantrone are used in treating cancer, in particular, in goodrisk patients. In the present method of the invention for treating oralleviating cancer, the cancer patient can be administered anti-FcRH5antibody in conjunction with treatment with the one or more of thepreceding chemotherapeutic agents. In particular, combination therapywith palictaxel and modified derivatives (see, e.g., EP0600517) iscontemplated. The anti-FcRH5 antibody will be administered with atherapeutically effective dose of the chemotherapeutic agent. In anotherembodiment, the anti-FcRH5 antibody is administered in conjunction withchemotherapy to enhance the activity and efficacy of thechemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference(PDR) discloses dosages of these agents that have been used in treatmentof various cancers. The dosing regimen and dosages of theseaforementioned chemotherapeutic drugs that are therapeutically effectivewill depend on the particular cancer being treated, the extent of thedisease and other factors familiar to the physician of skill in the artand can be determined by the physician.

In one particular embodiment, a conjugate comprising an anti-FcRH5antibody conjugated with a cytotoxic agent is administered to thepatient. Preferably, the immunoconjugate bound to the FcRH5 protein isinternalized by the cell, resulting in increased therapeutic efficacy ofthe immunoconjugate in killing the cancer cell to which it binds. In apreferred embodiment, the cytotoxic agent targets or interferes with thenucleic acid in the cancer cell. Examples of such cytotoxic agents aredescribed above and include maytansinoids, calicheamicins, ribonucleasesand DNA endonucleases.

The anti-FcRH5 antibodies or toxin conjugates thereof are administeredto a human patient, in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

An ex vivo strategy can also be used for therapeutic applications. Exvivo strategies involve transfecting or transducing cells obtained fromthe subject with a polynucleotide encoding a FcRH5 antagonist. Thetransfected or transduced cells are then returned to the subject. Thecells can be any of a wide range of types including, without limitation,hemopoietic cells (e.g., bone marrow cells, macrophages, monocytes,dendritic cells, T cells, or B cells), fibroblasts, epithelial cells,endothelial cells, keratinocytes, or muscle cells.

For example, if the FcRH5 antagonist is an antibody or immunoconjugate,the antagonist is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antagonist is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

In another example, the FcRH5 antagonist compound is administeredlocally, e.g., by direct injections, when the disorder or location ofthe tumor permits, and the injections can be repeated periodically. TheFcRH5 antagonist can also be delivered systemically to the subject ordirectly to the tumor cells, e.g., to a tumor or a tumor bed followingsurgical excision of the tumor, in order to prevent or reduce localrecurrence or metastasis.

Administration of the therapeutic agents in combination typically iscarried out over a defined time period (usually minutes, hours, days orweeks depending upon the combination selected). Combination therapy isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner.

The therapeutic agent can be administered by the same route or bydifferent routes. For example, the anti-FcRH antibody or immunoconjugatein the combination may be administered by intravenous injection while achemotherapeutic agent in the combination may be administered orally.Alternatively, for example, both of the therapeutic agents may beadministered orally, or both therapeutic agents may be administered byintravenous injection, depending on the specific therapeutic agents. Thesequence in which the therapeutic agents are administered also variesdepending on the specific agents.

Depending on the type and severity of the disease, about 1 μg/kg to 100mg/kg of each therapeutic agent is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until the cancer is treated, as measured by the methodsdescribed above. However, other dosage regimens may be useful.

The present application contemplates administration of the anti-FcRH5antibody by gene therapy. See, for example, WO96/07321 published Mar.14, 1996 concerning the use of gene therapy to generate intracellularantibodies.

Other therapeutic regimens may be combined with the administration ofthe anti-FcRH5 antibody. The combined administration includesco-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.Preferably such combined therapy results in a synergistic therapeuticeffect.

It may also be desirable to combine administration of the anti-FcRH5antibody or antibodies, with administration of an antibody directedagainst another tumor antigen associated with the particular cancer.

In another embodiment, the therapeutic treatment methods of the presentinvention involves the combined administration of an anti-FcRH5 antibody(or antibodies), and one or more chemotherapeutic agents or growthinhibitory agents, including co-administration of cocktails of differentchemotherapeutic agents, or other cytotoxic agent(s) or othertherapeutic agent(s) which also inhibits tumor growth. Chemotherapeuticagents include estramustine phosphate, prednimustine, cisplatin,5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea andhydroxyureataxanes (such as paclitaxel and doxetaxel) and/oranthracycline antibiotics. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992). The antibody may be combined with ananti-hormonal compound; e.g., an anti-estrogen compound such astamoxifen; an anti-progesterone such as onapristone (see, EP 616 812);or an anti-androgen such as flutamide, in dosages known for suchmolecules. Where the cancer to be treated is androgen independentcancer, the patient may previously have been subjected to anti-androgentherapy and, after the cancer becomes androgen independent, theanti-FcRH5 antibody (and optionally other agents as described herein)may be administered to the patient.

Sometimes, it may be beneficial to also co-administer a cardioprotectant(to prevent or reduce myocardial dysfunction associated with thetherapy) or one or more cytokines to the patient. In addition to theabove therapeutic regimes, the patient may be subjected to surgicalremoval of cancer cells and/or radiation therapy (e.g. external beamirradiation or therapy with a radioactive labeled agent, such as anantibody), before, simultaneously with, or post antibody therapy.Suitable dosages for any of the above co-administered agents are thosepresently used and may be lowered due to the combined action (synergy)of the agent and anti-FcRH5 antibody.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the dosage and mode ofadministration will be chosen by the physician according to knowncriteria. The appropriate dosage of antibody will depend on the type ofdisease to be treated, as defined above, the severity and course of thedisease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Preferably, the antibody isadministered by intravenous infusion or by subcutaneous injections.Depending on the type and severity of the disease, about 1 μg/kg toabout 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. A dosing regimen can comprise administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the anti-FcRH5 antibody. However, other dosageregimens may be useful. A typical daily dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. The progress of this therapy can be readilymonitored by conventional methods and assays and based on criteria knownto the physician or other persons of skill in the art.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of the antibody by genetherapy. Such administration of nucleic acid encoding the antibody isencompassed by the expression “administering a therapeutically effectiveamount of an antibody”. See, for example, WO96/07321 published Mar. 14,1996 concerning the use of gene therapy to generate intracellularantibodies.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells; in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the site where the antibody is required. For ex vivotreatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retroviral vector.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). For review of the currently known gene marking and genetherapy protocols see Anderson et al., Science 256:808-813 (1992). Seealso WO 93/25673 and the references cited therein.

The anti-FcRH5 antibodies of the invention can be in the different formsencompassed by the definition of “antibody” herein. Thus, the antibodiesinclude full length or intact antibody, antibody fragments, nativesequence antibody or amino acid variants, humanized, chimeric or fusionantibodies, immunoconjugates, and functional fragments thereof. Infusion antibodies an antibody sequence is fused to a heterologouspolypeptide sequence. The antibodies can be modified in the Fc region toprovide desired effector functions. As discussed in more detail in thesections herein, with the appropriate Fc regions, the naked antibodybound on the cell surface can induce cytotoxicity, e.g., viaantibody-dependent cellular cytotoxicity (ADCC) or by recruitingcomplement in complement dependent cytotoxicity, or some othermechanism. Alternatively, where it is desirable to eliminate or reduceeffector function, so as to minimize side effects or therapeuticcomplications, certain other Fc regions may be used.

In one embodiment, the antibody competes for binding or bindsubstantially to, the same epitope as the antibodies of the invention.Antibodies having the biological characteristics of the presentanti-FcRH5 antibodies of the invention are also contemplated,specifically including the in vivo tumor targeting and any cellproliferation inhibition or cytotoxic characteristics.

Methods of producing the above antibodies are described in detailherein.

The present anti-FcRH5 antibodies are useful for treating aFcRH5-expressing cancer or alleviating one or more symptoms of thecancer in a mammal. Such a cancer includes, but is not limited to,hematopoietic cancers or blood-related cancers, such as lymphoma,leukemia, myeloma or lymphoid malignancies, but also cancers of thespleen and cancers of the lymph nodes. More particular examples of suchB-cell associated cancers, including for example, high, intermediate andlow grade lymphomas (including B cell lymphomas such as, for example,mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin'slymphoma, mantle cell lymphoma, Burkitt's lymphoma, small lymphocyticlymphoma, marginal zone lymphoma, diffuse large cell lymphoma,follicular lymphoma, and Hodgkin's lymphoma and T cell lymphomas) andleukemias (including secondary leukemia, chronic lymphocytic leukemia,such as B cell leukemia (CD5+ B lymphocytes), myeloid leukemia, such asacute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia,such as acute lymphoblastic leukemia and myelodysplasia), and otherhematological and/or B cell- or T-cell-associated cancers. The cancersencompass metastatic cancers of any of the preceding. The antibody isable to bind to at least a portion of the cancer cells that expressFcRH5 polypeptide in the mammal. In a preferred embodiment, the antibodyis effective to destroy or kill FcRH5-expressing tumor cells or inhibitthe growth of such tumor cells, in vitro or in vivo, upon binding toFcRH5 polypeptide on the cell. Such an antibody includes a nakedanti-FcRH5 antibody (not conjugated to any agent). Naked antibodies thathave cytotoxic or cell growth inhibition properties can be furtherharnessed with a cytotoxic agent to render them even more potent intumor cell destruction. Cytotoxic properties can be conferred to ananti-FcRH5 antibody by, e.g., conjugating the antibody with a cytotoxicagent, to form an immunoconjugate as described herein. The cytotoxicagent or a growth inhibitory agent is preferably a small molecule.Toxins such as calicheamicin or a maytansinoid and analogs orderivatives thereof, are preferable.

The invention provides a composition comprising an anti-FcRH5 antibodyof the invention, and a carrier. For the purposes of treating cancer,compositions can be administered to the patient in need of suchtreatment, wherein the composition can comprise one or more anti-FcRH5antibodies present as an immunoconjugate or as the naked antibody. In afurther embodiment, the compositions can comprise these antibodies incombination with other therapeutic agents such as cytotoxic or growthinhibitory agents, including chemotherapeutic agents. The invention alsoprovides formulations comprising an anti-FcRH5 antibody of theinvention, and a carrier. In one embodiment, the formulation is atherapeutic formulation comprising a pharmaceutically acceptablecarrier.

Another aspect of the invention is isolated nucleic acids encoding theanti-FcRH5 antibodies. Nucleic acids encoding both the H and L chainsand especially the hypervariable region residues, chains which encodethe native sequence antibody as well as variants, modifications andhumanized versions of the antibody, are encompassed.

The invention also provides methods useful for treating a FcRH5polypeptide-expressing cancer or alleviating one or more symptoms of thecancer in a mammal, comprising administering a therapeutically effectiveamount of an anti-FcRH5 antibody to the mammal. The antibody therapeuticcompositions can be administered short term (acute) or chronic, orintermittent as directed by physician. Also provided are methods ofinhibiting the growth of, and killing a FcRH5 polypeptide-expressingcell.

The invention also provides kits and articles of manufacture comprisingat least one anti-FcRH5 antibody. Kits containing anti-FcRH5 antibodiesfind use, e.g., for FcRH5 cell killing assays, for purification orimmunoprecipitation of FcRH5 polypeptide from cells. For example, forisolation and purification of FcRH5, the kit can contain an anti-FcRH5antibody coupled to beads (e.g., sepharose beads). Kits can be providedwhich contain the antibodies for detection and quantitation of FcRH5 invitro, e.g., in an ELISA or a Western blot. Such antibody useful fordetection may be provided with a label such as a fluorescent orradiolabel.

I. Antibody-Drug Conjugate Treatments

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia and lymphoid malignancies. Others include neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologic,including autoimmune, disorders.

The ADC compounds which are identified in the animal models andcell-based assays can be further tested in tumor-bearing higher primatesand human clinical trials. Human clinical trials can be designed to testthe efficacy of the anti-FcRH5 monoclonal antibody or immunoconjugate ofthe invetion in patients experiencing a B cell proliferative disorderincluding without limitation lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma. The clinicaltrial may be designed to evaluate the efficacy of an ADC in combinationswith known therapeutic regimens, such as radiation and/or chemotherapyinvolving known chemotherapeutic and/or cytotoxic agents.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as a B cell proliferative disorder and/or a B cell cancer.Examples of cancer to be treated herein include, but are not limited to,B cell proliferative disorder is selected from lymphoma, non-Hodgkinslymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsedindolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

The cancer may comprise FcRH5-expressing cells, such that the ADC of thepresent invention are able to bind to the cancer cells. To determineFcRH5 expression in the cancer, various diagnostic/prognostic assays areavailable. In one embodiment, FcRH5 overexpression may be analyzed byIHC. Parrafin-embedded tissue sections from a tumor biopsy may besubjected to the IHC assay and accorded a FcRH5 protein stainingintensity criteria with respect to the degree of staining and in whatproportion of tumor cells examined.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of an anti-ErbB2 antibody.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

J. Combination Therapy

An antibody-drug conjugate (ADC) of the invention may be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen preferably has complementary activities to the ADC of thecombination such that they do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended. A pharmaceuticalcomposition containing an ADC of the invention may also have atherapeutically effective amount of a chemotherapeutic agent such as atubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

In one aspect, the first compound is an anti-FcRH5 ADC of the inventionand the second compound is an anti-CD20 antibody (either a nakedantibody or an ADC). In one embodiment the second compound is ananti-CD20 antibody rituximab (Rituxan®) or 2H7 (Genentech, Inc., SouthSan Francisco, Calif.). Another antibodies useful for combinedimmunotherapy with anti-FcRH5 ADCs of the invention includes withoutlimitation, anti-VEGF (e.g, Avastin®).

Other therapeutic regimens may be combined with the administration of ananticancer agent identified in accordance with this invention, includingwithout limitation radiation therapy and/or bone marrow and peripheralblood transplants, and/or a cytotoxic agent, a chemotherapeutic agent,or a growth inhibitory agent. In one of such embodiments, achemotherapeutic agent is an agent or a combination of agents such as,for example, cyclophosphamide, hydroxydaunorubicin, adriamycin,doxorubincin, vincristine (Oncovin™) prednisolone, CHOP, CVP, or COP, orimmunotherapeutics such as anti-CD20 (e.g., Rituxan®) or anti-VEGF(e.g., Avastin®).

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

In one embodiment, treatment with an ADC involves the combinedadministration of an anticancer agent identified herein, and one or morechemotherapeutic agents or growth inhibitory agents, includingcoadministration of cocktails of different chemotherapeutic agents.Chemotherapeutic agents include taxanes (such as paclitaxel anddocetaxel) and/or anthracycline antibiotics. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturer's instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in “Chemotherapy Service”, (1992) Ed., M. C. Perry,Williams & Wilkins, Baltimore, Md.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

The combination therapy of the invention can further comprise one ormore chemotherapeutic agent(s). The combined administration includescoadministration or concurrent administration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities.

The chemotherapeutic agent, if administered, is usually administered atdosages known therefor, or optionally lowered due to combined action ofthe drugs or negative side effects attributable to administration of theantimetabolite chemotherapeutic agent. Preparation and dosing schedulesfor such chemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.

Various chemotherapeutic agents that can be combined are disclosedherein.

In some embodiments, chemotherapeutic agents to be combined are selectedfrom the group consisting of immunomodulators (IMids) (includingthalidomide and Revlimid® (lenalidomide)), proteosome inhibitors (suchas Velcade® (bortezomib) and PS342), bora taxoid (including docetaxeland paclitaxel), vinca (such as vinorelbine or vinblastine), platinumcompound (such as carboplatin or cisplatin), aromatase inhibitor (suchas letrozole, anastrazole, or exemestane), anti-estrogen (e.g.fulvestrant or tamoxifen), etoposide, thiotepa, cyclophosphamide,pemetrexed, methotrexate, liposomal doxorubicin, pegylated liposomaldoxorubicin, capecitabine, gemcitabine, melthalin, doxorubicin,vincristine, COX-2 inhibitor (for instance, celecoxib), or steroid(e.g., dexamethasone and prednisone). In some embodiments (e.g.,embodiments involving treatment of multiple myeloma), dexamethasone andlenalidomide, or dexamethasone, or bortezomib, or vincristine,doxorubicin and dexamethason, or thalidomide and dexamethasone, orliposomal doxorubicin, vincristine and dexamethasone, or lenalidomideand dexamethasone, or bortezomib and dexamethasone, or bortezomib,doxorubicin, and dexamethasone are combined.

In some embodiments, chemotherapeutic agents to be combined are selectedfrom the group consisting of Revlimid® (lenalidomide), proteosomeinhibitors (such as Velcade® (bortezomib) and PS342), bora taxoid(including docetaxel and paclitaxel), vinca (such as vinorelbine orvinblastine), platinum compound (such as carboplatin or cisplatin),aromatase inhibitor (such as letrozole, anastrazole, or exemestane),anti-estrogen (e.g. fulvestrant or tamoxifen), etoposide, thiotepa,cyclophosphamide, pemetrexed, methotrexate, liposomal doxorubicin,pegylated liposomal doxorubicin, capecitabine, gemcitabine, melthalin,doxorubicin, vincristine, COX-2 inhibitor (for instance, celecoxib), orsteroid (e.g., dexamethasone and prednisone).

In some embodiments (e.g., embodiments involving treatment of malignantmyeloma), dexamethasone and lenalidomide, or dexamethasone, andbortezomib are combined.

In one other embodiment, the combination therapy comprises an antibodyor immunoconjugate and more than one chemotherapeutic agent. Forexample, the antibody or immunoconjugate may be combined with (i)vincristine, doxorubicin (in a liposomal or non-liposomal formulation),and dexamethasone; (ii) thalidomide and dexamethasone; (iii) Velcade®(bortezomib), doxorubicin, and dexamethasone; (iv) Velcade®(bortezomib), thalidomide, and dexamethasone; (v) melphalan andprednisone; (vi) melphalan, prednisone, and thalidomide; (vii)melphalan, prednisone, and Velcade® (bortezomib); (viii) vincristine,doxorubicin, and dexamethasone; or (ix) thalidomide and dexamethasone.

In one embodiment, the combination therapy may comprise an antibody orimmunoconjugate described herein and one or more of (i) Velcade®(bortezomib), (ii) dexamethasone, (iii) Revlimid® (lenalidomide). Inanother embodiment, the combination therapy comprises an antibody orimmunoconjugate and both dexamethasone and Revlimid® (lenalidomide) orboth Velcade® (bortezomib) and dexamethasone. In one other embodiment,the combination therapy comprises an antibody or immunoconjugate andeach of Velcade® (bortezomib), dexamethasone and Revlimid®(lenalidomide).

K. Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of FcRH5-expressing cancer. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating, preventing and/or diagnosing the cancercondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an anti-FcRH5 antibody of the invention. The label orpackage insert indicates that the composition is used for treatingcancer. The label or package insert will further comprise instructionsfor administering the antibody composition to the cancer patient.Additionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, e.g., forFcRH5-expressing cell killing assays, for purification orimmunoprecipitation of FcRH5 polypeptide from cells. For isolation andpurification of FcRH5 polypeptide, the kit can contain an anti-FcRH5antibody coupled to beads (e.g., sepharose beads). Kits can be providedwhich contain the antibodies for detection and quantitation of FcRH5polypeptide in vitro, e.g., in an ELISA or a Western blot. As with thearticle of manufacture, the kit comprises a container and a label orpackage insert on or associated with the container. The container holdsa composition comprising at least one anti-FcRH5 antibody of theinvention. Additional containers may be included that contain, e.g.,diluents and buffers, control antibodies. The label or package insertmay provide a description of the composition as well as instructions forthe intended in vitro or detection use.

L. Uses for FcRH5 Polypeptides

This invention encompasses methods of screening compounds to identifythose that mimic the FcRH5 polypeptide (agonists) or prevent the effectof the FcRH5 polypeptide (antagonists). Screening assays for antagonistdrug candidates are designed to identify compounds that bind or complexwith the FcRH5 polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins, including e.g., inhibiting the expressionof FcRH5 polypeptide from cells. Such screening assays will includeassays amenable to high-throughput screening of chemical libraries,making them particularly suitable for identifying small molecule drugcandidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a FcRH5 polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the FcRH5 polypeptide encoded by the gene identified hereinor the drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the FcRH5 polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for theFcRH5 polypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular FcRH5 polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a FcRH5polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the FcRH5 polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the FcRH5 polypeptide indicates that the compound is anantagonist to the FcRH5 polypeptide. Alternatively, antagonists may bedetected by combining the FcRH5 polypeptide and a potential antagonistwith membrane-bound FcRH5 polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. TheFcRH5 polypeptide can be labeled, such as by radioactivity, such thatthe number of FcRH5 polypeptide molecules bound to the receptor can beused to determine the effectiveness of the potential antagonist. Thegene encoding the receptor can be identified by numerous methods knownto those of skill in the art, for example, ligand panning and FACSsorting. Coligan et al., Current Protocols in Immun, 1(2): Chapter 5(1991). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to the FcRH5polypeptide and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to the FcRH5 polypeptide. Transfected cells that are grown onglass slides are exposed to labeled FcRH5 polypeptide. The FcRH5polypeptide can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an interactive sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor.

As an alternative approach for receptor identification, labeled FcRH5polypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledFcRH5 polypeptide in the presence of the candidate compound. The abilityof the compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with FcRH5polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of theFcRH5 polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the FcRH5 polypeptide.

Antibodies specifically binding a FcRH5 polypeptide identified herein,as well as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of variousdisorders, including cancer, in the form of pharmaceutical compositions.

If the FcRH5 polypeptide is intracellular and whole antibodies are usedas inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

M. Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

N. Method of Screening

Yet another embodiment of the present invention is directed to a methodof determining the presence of a FcRH5 polypeptide in a sample suspectedof containing the FcRH5 polypeptide, wherein the method comprisesexposing the sample to an antibody drug conjugate thereof, that binds tothe FcRH5 polypeptide and determining binding of the antibody drugconjugate thereof, to the FcRH5 polypeptide in the sample, wherein thepresence of such binding is indicative of the presence of the FcRH5polypeptide in the sample. Optionally, the sample may contain cells(which may be cancer cells) suspected of expressing the FcRH5polypeptide. The antibody drug conjugate thereof, employed in the methodmay optionally be detectably labeled, attached to a solid support, orthe like.

Another embodiment of the present invention is directed to a method ofdiagnosing the presence of a tumor in a mammal, wherein the methodcomprises (a) contacting a test sample comprising tissue cells obtainedfrom the mammal with an antibody drug conjugate thereof, that binds to aFcRH5 polypeptide and (b) detecting the formation of a complex betweenthe antibody drug conjugate thereof, and the FcRH5 polypeptide in thetest sample, wherein the formation of a complex is indicative of thepresence of a tumor in the mammal. Optionally, the antibody drugconjugate thereof, is detectably labeled, attached to a solid support,or the like, and/or the test sample of tissue cells is obtained from anindividual suspected of having a cancerous tumor.

IV. FURTHER METHODS OF USING ANTI-FCRH5 ANTIBODIES AND IMMUNOCONJUGATES

A. Diagnostic Methods and Methods of Detection

In one aspect, anti-FcRH5 antibodies and immunoconjugates of theinvention are useful for detecting the presence of FcRH5 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue. In certain embodiments, such tissues includenormal and/or cancerous tissues that express FcRH5 at higher levelsrelative to other tissues, for example, B cells and/or B cell associatedtissues.

In one aspect, the invention provides a method of detecting the presenceof FcRH5 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-FcRH5 antibodyunder conditions permissive for binding of the anti-FcRH5 antibody toFcRH5, and detecting whether a complex is formed between the anti-FcRH5antibody and FcRH5.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of FcRH5. In certain embodiments,the method comprises contacting a test cell with an anti-FcRH5 antibody;determining the level of expression (either quantitatively orqualitatively) of FcRH5 by the test cell by detecting binding of theanti-FcRH5 antibody to FcRH5; and comparing the level of expression ofFcRH5 by the test cell with the level of expression of FcRH5 by acontrol cell (e.g., a normal cell of the same tissue origin as the testcell or a cell that expresses FcRH5 at levels comparable to such anormal cell), wherein a higher level of expression of FcRH5 by the testcell as compared to the control cell indicates the presence of adisorder associated with increased expression of FcRH5. In certainembodiments, the test cell is obtained from an individual suspected ofhaving a disorder associated with increased expression of FcRH5. Incertain embodiments, the disorder is a cell proliferative disorder, suchas a cancer or a tumor.

Exemplary cell proliferative disorders that may be diagnosed using anantibody of the invention include a B cell disorder and/or a B cellproliferative disorder including, but not limited to, lymphoma,non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL, chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairycell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle celllymphoma.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-FcRH5antibody to FcRH5 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing FcRH5 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-FcRH5 antibody under conditions permissive for binding of theanti-FcRH5 antibody to FcRH5, and detecting whether a complex is formedbetween the anti-FcRH5 antibody and FcRH5 on the cell surface. Anexemplary assay for detecting binding of an anti-FcRH5 antibody to FcRH5expressed on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-FcRH5antibodies to FcRH5. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-FcRH5 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In certain embodiments, anti-FcRH5 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-FcRH5antibody from any FcRH5 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-FcRH5antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-FcRH5 antibody afterformation of a complex between the anti-FcRH5 antibody and FcRH5, e.g.,by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-FcRH5 antibody.

B. Therapeutic Methods

An antibody or immunoconjugate of the invention may be used in, forexample, in vitro, ex vivo, and in vivo therapeutic methods. In oneaspect, the invention provides methods for inhibiting cell growth orproliferation, either in vivo or in vitro, the method comprisingexposing a cell to an anti-FcRH5 antibody or immunoconjugate thereofunder conditions permissive for binding of the immunoconjugate to FcRH5.“Inhibiting cell growth or proliferation” means decreasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, and includes inducing cell death. In certainembodiments, the cell is a tumor cell. In certain embodiments, the cellis a B cell. In certain embodiments, the cell is a xenograft, e.g., asexemplified herein.

In one aspect, an antibody or immunoconjugate of the invention is usedto treat or prevent a B cell proliferative disorder. In certainembodiments, the cell proliferative disorder is associated withincreased expression and/or activity of FcRH5. For example, in certainembodiments, the B cell proliferative disorder is associated withincreased expression of FcRH5 on the surface of a B cell. In certainembodiments, the B cell proliferative disorder is a tumor or a cancer.Examples of B cell proliferative disorders to be treated by theantibodies or immunoconjugates of the invention include, but are notlimited to, lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL,relapsed aggressive NHL, relapsed indolent NHL, refractory NHL,refractory indolent NHL, chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acutelymphocytic leukemia (ALL), and mantle cell lymphoma.

In one aspect, the invention provides methods for treating a B cellproliferative disorder comprising administering to an individual aneffective amount of an anti-FcRH5 antibody or immunoconjugate thereof.In certain embodiments, a method for treating a B cell proliferativedisorder comprises administering to an individual an effective amount ofa pharmaceutical formulation comprising an anti-FcRH5 antibody oranti-FcRH5 immunoconjugate and, optionally, at least one additionaltherapeutic agent, such as those provided below. In certain embodiments,a method for treating a cell proliferative disorder comprisesadministering to an individual an effective amount of a pharmaceuticalformulation comprising 1) an immunoconjugate comprising an anti-FcRH5antibody and a cytotoxic agent; and optionally, 2) at least oneadditional therapeutic agent, such as those provided below.

In one aspect, at least some of the antibodies or immunoconjugates ofthe invention can bind FcRH5 from species other than human. Accordingly,antibodies or immunoconjugates of the invention can be used to bindFcRH5, e.g., in a cell culture containing FcRH5, in humans, or in othermammals having a FcRH5 with which an antibody or immunoconjugate of theinvention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgusand rhesus monkeys, pig or mouse). In one embodiment, an anti-FcRH5antibody or immunoconjugate can be used for targeting FcRH5 on B cellsby contacting the antibody or immunoconjugate with FcRH5 to form anantibody or immunoconjugate-antigen complex such that a conjugatedcytotoxin of the immunoconjugate accesses the interior of the cell. Inone embodiment, the FcRH5 is human FcRH5.

In one embodiment, an anti-FcRH5 antibody or immunoconjugate can be usedin a method for binding FcRH5 in an individual suffering from a disorderassociated with increased FcRH5 expression and/or activity, the methodcomprising administering to the individual the antibody orimmunoconjugate such that FcRH5 in the individual is bound. In oneembodiment, the bound antibody or immunoconjugate is internalized intothe B cell expressing FcRH5. In one embodiment, the FcRH5 is humanFcRH5, and the individual is a human individual. Alternatively, theindividual can be a mammal expressing FcRH5 to which an anti-FcRH5antibody binds. Still further the individual can be a mammal into whichFcRH5 has been introduced (e.g., by administration of FcRH5 or byexpression of a transgene encoding FcRH5).

An anti-FcRH5 antibody or immunoconjugate can be administered to a humanfor therapeutic purposes. Moreover, an anti-FcRH5 antibody orimmunoconjugate can be administered to a non-human mammal expressingFcRH5 with which the antibody cross-reacts (e.g., a primate, pig, rat,or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies or immunoconjugates ofthe invention (e.g., testing of dosages and time courses ofadministration).

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other compositions in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent and/or adjuvant. In certainembodiments, an additional therapeutic agent is a cytotoxic agent, achemotherapeutic agent, or a growth inhibitory agent. In one of suchembodiments, a chemotherapeutic agent is an agent or a combination ofagents such as, for example, cyclophosphamide, hydroxydaunorubicin,adriamycin, doxorubincin, vincristine (Oncovin™), prednisolone, CHOP,CVP, or COP, or immunotherapeutics such as anti-CD20 (e.g., Rituxan®) oranti-VEGF (e.g., Avastin®), wherein the combination therapy is useful inthe treatment of cancers and/or B cell disorders such as B cellproliferative disorders including lymphoma, non-Hodgkins lymphoma (NHL),aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL),acute lymphocytic leukemia (ALL), and mantle cell lymphoma.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent or adjuvant) can be administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody orimmunoconjugate is suitably administered by pulse infusion, particularlywith declining doses of the antibody or immunoconjugate. Dosing can beby any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents, suchas chemotherapeutic agents) will depend on the type of disease to betreated, the type of antibody or immunoconjugate, the severity andcourse of the disease, whether the antibody or immunoconjugate isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody orimmunoconjugate, and the discretion of the attending physician. Theantibody or immunoconjugate is suitably administered to the patient atone time or over a series of treatments. Depending on the type andseverity of the disease, about 1 μg/kg to 100 mg/kg (e.g. 0.1 mg/kg-20mg/kg) of antibody or immunoconjugate can be an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody or immunoconjugate would bein the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or moredoses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or anycombination thereof) of antibody or immunoconjugate may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of the antibodyor immunoconjugate). An initial higher loading dose, followed by one ormore lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the antibody.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

C. Activity Assays

Anti-FcRH5 antibodies and immunoconjugates of the invention may becharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Activity Assays

In one aspect, assays are provided for identifying anti-FcRH5 antibodiesor immunoconjugates thereof having biological activity. Biologicalactivity may include, e.g., the ability to inhibit cell growth orproliferation (e.g., “cell killing” activity), or the ability to inducecell death, including programmed cell death (apoptosis). Antibodies orimmunoconjugates having such biological activity in vivo and/or in vitroare also provided.

In certain embodiments, an anti-FcRH5 antibody or immunoconjugatethereof is tested for its ability to inhibit cell growth orproliferation in vitro. Assays for inhibition of cell growth orproliferation are well known in the art. Certain assays for cellproliferation, exemplified by the “cell killing” assays describedherein, measure cell viability. One such assay is the CellTiter-Glo™Luminescent Cell Viability Assay, which is commercially available fromPromega (Madison, Wis.). That assay determines the number of viablecells in culture based on quantitation of ATP present, which is anindication of metabolically active cells. See Crouch et al (1993) J.Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

Another assay for cell proliferation is the “MTT” assay, a colorimetricassay that measures the oxidation of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazanby mitochondrial reductase. Like the CellTiter-Glo™ assay, this assayindicates the number of metabolically active cells present in a cellculture. See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhanget al. (2005) Cancer Res. 65:3877-3882.

In one aspect, an anti-FcRH5 antibody is tested for its ability toinduce cell death in vitro. Assays for induction of cell death are wellknown in the art. In some embodiments, such assays measure, e.g., lossof membrane integrity as indicated by uptake of propidium iodide (PI),trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD.In an exemplary PI uptake assay, cells are cultured in Dulbecco'sModified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay isperformed in the absence of complement and immune effector cells. Cellsare seeded at a density of 3×10⁶ per dish in 100×20 mm dishes andallowed to attach overnight. The medium is removed and replaced withfresh medium alone or medium containing various concentrations of theantibody or immunoconjugate. The cells are incubated for a 3-day timeperiod. Following treatment, monolayers are washed with PBS and detachedby trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutesat 4° C., the pellet resuspended in 3 ml cold Ca²⁺ binding buffer (10 mMHepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 mm tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Antibodies or immunoconjugateswhich induce statistically significant levels of cell death asdetermined by PI uptake are thus identified.

In one aspect, an anti-FcRH5 antibody or immunoconjugate is tested forits ability to induce apoptosis (programmed cell death) in vitro. Anexemplary assay for antibodies or immunconjugates that induce apoptosisis an annexin binding assay. In an exemplary annexin binding assay,cells are cultured and seeded in dishes as discussed in the precedingparagraph. The medium is removed and replaced with fresh medium alone ormedium containing 0.001 to 10 μg/ml of the antibody or immunoconjugate.Following a three-day incubation period, monolayers are washed with PBSand detached by trypsinization. Cells are then centrifuged, resuspendedin Ca²⁺ binding buffer, and aliquoted into tubes as discussed in thepreceding paragraph. Tubes then receive labeled annexin (e.g. annexinV-FITC) (1 μg/ml). Samples are analyzed using a FACSCAN™ flow cytometerand FACSCONVERT™ CellQuest software (BD Biosciences). Antibodies orimmunoconjugates that induce statistically significant levels of annexinbinding relative to control are thus identified. Another exemplary assayfor antibodies or immunconjugates that induce apoptosis is a histone DNAELISA colorimetric assay for detecting internucleosomal degradation ofgenomic DNA. Such an assay can be performed using, e.g., the Cell DeathDetection ELISA kit (Roche, Palo Alto, Calif.).

Cells for use in any of the above in vitro assays include cells or celllines that naturally express FcRH5 or that have been engineered toexpress FcRH5. Such cells include tumor cells that overexpress FcRH5relative to normal cells of the same tissue origin. Such cells alsoinclude cell lines (including tumor cell lines) that express FcRH5 andcell lines that do not normally express FcRH5 but have been transfectedwith nucleic acid encoding FcRH5.

In one aspect, an anti-FcRH5 antibody or immunoconjugate thereof istested for its ability to inhibit cell growth or proliferation in vivo.In certain embodiments, an anti-FcRH5 antibody or immunoconjugatethereof is tested for its ability to inhibit tumor growth in vivo. Invivo model systems, such as xenograft models, can be used for suchtesting. In an exemplary xenograft system, human tumor cells areintroduced into a suitably immunocompromised non-human animal, e.g., aSCID mouse. An antibody or immunoconjugate of the invention isadministered to the animal. The ability of the antibody orimmunoconjugate to inhibit or decrease tumor growth is measured. Incertain embodiments of the above xenograft system, the human tumor cellsare tumor cells from a human patient. Such cells useful for preparingxenograft models include human leukemia and lymphoma cell lines, whichinclude without limitation the BJAB-luc cells (an EBV-negative Burkitt'slymphoma cell line transfected with the luciferase reporter gene), Ramoscells (ATCC, Manassas, Va., CRL-1923), SuDHL-4 cells (DSMZ,Braunschweig, Germany, AAC 495), DoHH2 cells (see Kluin-Neilemans, H. C.et al., Leukemia 5:221-224 (1991), and Kluin-Neilemans, H. C. et al.,Leukemia 8:1385-1391 (1994)), Granta-519 cells (see Jadayel, D. M. etal, Leukemia 11(1):64-72 (1997)). In certain embodiments, the humantumor cells are introduced into a suitably immunocompromised non-humananimal by subcutaneous injection or by transplantation into a suitablesite, such as a mammary fat pad.

2. Binding Assays and Other Assays

In one aspect, an anti-FcRH5 antibody is tested for its antigen bindingactivity. For example, in certain embodiments, an anti-FcRH5 antibody istested for its ability to bind to FcRH5 expressed on the surface of acell. A FACS assay may be used for such testing.

In one aspect, competition assays may be used to identify a monoclonalantibody that competes with murine 13G9 antibody (mu13G9), humanized13G9 antibody and/or humanized 13G9.v1 and/or humanized 13G9.v3 and/orhumanized 13G9.v8 antibody for binding to FcRH5. In certain embodiments,such a competing antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by murine 13G9 antibody, humanized13G9.v1 antibody and/or humanized 13G9.v3 antibody and/or humanized13G9.v8. Exemplary competition assays include, but are not limited to,routine assays such as those provided in Harlow and Lane (1988)Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.). Two antibodies are said to bind to thesame epitope if each blocks binding of the other by 50% or more.

In an exemplary competition assay, immobilized FcRH5 is incubated in asolution comprising a first labeled antibody that binds to FcRH5 (e.g.,murine 13G9 (mu13G9) antibody, chimeric 13G9 (ch13G9), and/or humanized13G9 (hu13G9) antibody) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toFcRH5. The second antibody may be present in a hybridoma supernatant. Asa control, immobilized FcRH5 is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto FcRH5, excess unbound antibody is removed, and the amount of labelassociated with immobilized FcRH5 is measured. If the amount of labelassociated with immobilized FcRH5 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toFcRH5. In certain embodiments, immobilized FcRH5 is present on thesurface of a cell or in a membrane preparation obtained from a cellexpressing FcRH5 on its surface.

In one aspect, purified anti-FcRH5 antibodies can be furthercharacterized by a series of assays including, but not limited to,N-terminal sequencing, amino acid analysis, non-denaturing sizeexclusion high pressure liquid chromatography (HPLC), mass spectrometry,ion exchange chromatography and papain digestion.

In one embodiment, the invention contemplates an altered antibody thatpossesses some but not all effector functions, which make it a desirablecandidate for many applications in which the half life of the antibodyin vivo is important yet certain effector functions (such as complementand ADCC) are unnecessary or deleterious. In certain embodiments, the Fcactivities of the antibody are measured to ensure that only the desiredproperties are maintained. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express Fc(RIII only, whereasmonocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).Clq binding assays may also be carried out to confirm that the antibodyis unable to bind Clq and hence lacks CDC activity. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed. FcRn binding and invivo clearance/half life determinations can also be performed usingmethods known in the art.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated.Antibodies used in the examples are commercially available antibodies orantibodies described herein. The source of those cells identified in thefollowing examples, and throughout the specification, by ATCC accessionnumbers, is the American Type Culture Collection, Manassas, Va.

Example 1 Preparation of Antibodies that Bind FcRH5

This example illustrates preparation of monoclonal antibodies which canspecifically bind FcRH5.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified FcRH5, fusion proteins containing FcRH5,and cells expressing recombinant FcRH5 on the cell surface. Selection ofthe immunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the FcRH5 immunogen emulsifiedin complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-FcRH5 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of immunogen. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstimmunogen. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against immunogen is within the skill inthe art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-immunogenmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Antibodies directed against the FcRH5 polypeptide disclosed herein canbe successfully produced using this technique(s). More specifically,functional monoclonal antibodies that are capable of recognizing andbinding to FcRH5 protein (as measured by standard ELISA, FACS sortinganalysis and/or immunohistochemistry analysis) can be successfullygenerated against the following FcRH5 protein as disclosed herein:PRO820 (DNA56041), PRO52387 (DNA257845).

In addition to the preparation of monoclonal antibodies directed againstthe FcRH5 polypeptides as described herein, many of the monoclonalantibodies can be successfully conjugated to a cell toxin for use indirecting the cellular toxin to a cell (or tissue) that expresses aFcRH5 polypeptide of interested (both in vitro and in vivo). Forexample, toxin (e.g., DM1) derivitized monoclonal antibodies can besuccessfully generated to the following FcRH5 polypeptides as describedherein: PRO820 (DNA56041), PRO52387 (DNA257845).

A. Generation of FcRH5/IRTA2 (PRO820, PRO52387) Stable Cell Lines

To make cell lines for screening the FcRH5 antibodies, SVT2 mousefibroblast cell lines stably expressing the tagged and untaggedFcRH5/IRTA2 were generated. The FcRH5/IRTA2 cDNA was PCR amplified froma spleen cell library and TA cloned (Invitrogen) into pCR4. To make theuntagged expression construct, the open reading frames (ORFs) werecloned into the mammalian expression vector pCMV.PD.nbe by using PCR toadd restriction sites, digestion of the PCR product and ligation intothe vector. N-terminal tagged expression constructs were made byamplification of the FcRH5/IRTA2 ORFs without the signal sequence andligation of the PCR product into the pMSCVneo (Clontech) vectorcontaining the gD tag and signal sequence (M G G T A A R L G A V I L F VV I V G H G V R G K Y A L A D A S L K M A D P N R F R G K D L P V L D QL L) (SEQ ID NO: 1). For each FcRH5/IRTA2, two stable cell lines wereestablished for use in screening the monoclonal antibodies for FACSspecific reactivity and crossreactivity between the FcRH/IRTAs. ThegD-tagged and untagged expression vectors were transfected into SVT2(grown in high glucose DMEM+10% FBS+2 mM L-glutamine cells) by thestandard Lipofectamine 2000 (Invitrogen; Carlsbad, Calif.) protocol. ThegD-tagged transfectants were put under 0.5 mg/ml Geneticin (Invitrogen;Carlsbad, Calif.) selection for one week and then single cell FACSsorted with gD-tag specific monoclonal antibody (gD:952, Genentech;South San Francisco) to acquire the highest expressing clone. Theuntagged transfectants were put under 5.0 ug/ml puromycin (Calbiochem;La Jolla, Calif.) selection until visible colonies grew out. RNA fromeach colony was isolated by the standard Trizolâ (Invitrogen; Carlsbad,Calif.) protocol and TaqManâ (ABI; Foster City, Calif.) run to determinethe highest producing clone.

B. Generation of Monoclonal Antibodies to FcRH5/IRTA2 (PRO820, PRO52387)

Murine monoclonal antibodies capable of binding human FcRH5 wereprepared.

Protein for immunization of mice was generated by transient transfectionof vectors that expresses the His-tagged extra-cellular domain (ECDs) ofthe FcRH5/IRTA2 into CHO cells. The protein was purified from thetransfected cell supernatants on nickel columns and the identity of theprotein confirmed by N-terminal sequencing.

Ten Balb/c mice (Charles River Laboratories, Hollister, Calif.) werehyperimmunized with recombinant polyhistidine-tagged (HIS6 (SEQ ID NO:69)) protein, in Ribi adjuvant (Ribi Immunochem Research, Inc.,Hamilton, Mo.). B-cells from mice demonstrating high antibody titersagainst the immunogen by direct ELISA, and specific binding to SVT2mouse fibroblast cells stably expressing the FcRH of interest by FACS,were fused with mouse myeloma cells (X63.Ag8.653; American Type CultureCollection, Rockville, Md.) using a modified protocol analogous to onepreviously described (Kohler and Milstein, 1975; Hongo et al., 1995).After 10-12 days, the supernatants were harvested and screened forantibody production and binding by direct ELISA and FACS. Positiveclones, showing the highest immunobinding after the second round ofsubcloning by limiting dilution, were expanded and cultured for furthercharacterization, including FcRH1, -2, -3, -4, and -5 specificity andcrossreactivity. The supernatants harvested from each hybridoma lineagewere purified by affinity chromatography (Pharmacia fast protein liquidchromatography [FPLC]; Pharmacia, Uppsala, Sweden) using a modifiedprotocol analogous to one previously described (Hongo et al., 1995). Thepurified antibody preparations were then sterile filtered (0.2-μm poresize; Nalgene, Rochester N.Y.) and stored at 4° C. in phosphate bufferedsaline (PBS).

Monoclonal antibodies that are capable of recognizing and binding toFcRH5 protein (as measured by standard ELISA, FACS sorting analysisand/or immunohistochemistry analysis) have been successfully generatedagainst the PRO52837 (FcRH5c/IRTA2c) and have been designated as:

anti-FcRH5-9E2 (herein referred to as 9E2 or 9E2.1.1) (ATCC No.PTA-7207, deposited on Nov. 9, 2005),

anti-FcRH5-1H4 (herein referred to as 1H4 or 1H4.1.1) (ATCC No.PTA-7208, deposited on Nov. 9, 2005),

anti-FcRH5-13G9 (herein referred to as 13G9 or 13G9.1.1) (describedherein), anti-FcRH5-4D10 (herein referred to as 4D10 or 4D10.1.1) (ATCCNo. PTA-7210, deposited on Nov. 9, 2005),

anti-FcRH5-6H1 (herein referred to as 6H1 or 6H1.1.1) (ATCC No.PTA-7211, deposited on Nov. 9, 2005),

anti-FcRH5-8H6 (herein referred to as 8H6 or 8H6.1.1) (ATCC No.PTA-7212, deposited on Nov. 9, 2005),

anti-FcRH5-7D11 (herein referred to as 7D11 or 7D11.1.1) (describedherein),

Anti-FCRH5-10A8 (herein referred to as 10A8 or 10A81.1) (described inU.S. Provisional Patent Application Nos. 61/211,695 filed Apr. 1, 2009,61/166,217 filed Apr. 2, 2009, and 61/266,972 filed Dec. 4, 2009, and aU.S. Non-Provisional application Ser. No. 12/751,819 filed on Mar. 31,2010, now issued as U.S. Pat. No. 8,466,260 on Jun. 18, 2013, each ofwhich is incorporated herein by reference in its entirety.))

1. Affinity Determination (Biacore Analysis)

The FcRH-binding affinities of IgGs were calculated from association anddissociation rate constants measured using a Biacore 2000 surfaceplasmon resonance system (BIAcore, Inc.). A biosensor chip was activatedfor covalent coupling of either anti-murine IgG (BR-1008-338) usingN-ethyl-N′-(3-dimethyaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instruction(BIAcore, Inc.). Briefly, for each cycle of kinetic analysis, 5 ul of100 nM IgG was captured on the chip, two-fold serial dilution of variousFcRHs were injected at a flow rate of 20 ul/min for 150 sec. Antigendissociation was monitored for 180 sec before regeneration. Equilibriumdissociation constants at 25° C. were calculated by fitting the datawith a 1:1 Langmuir binding model. Running buffer was PBS with 0.05%Tween −20. The results are shown below in Table 9. All the antibodiesbound antigen with sufficient affinity to be used as a therapeutic.

TABLE 9 Cyno X-reactivity Affinity (FACS) Murine (Biacore) on stabletransfected Antibody to Hu antigen cells 1H4 6 nM X* 4D10 3 nM 6H1 <0.1nM X 7D11 6 nM X* 8H6 20 nM X (by IHC) 9E2 0.2 nM 10A8 N/A X 10F5 N/A X13G9 19 nM X *Very slight at saturating concentration

C. Construction and Sequencing of Chimeric Anti-Human FcRH5 Antibodies

1. Chimeric Anti-Human FcRH5 Antibody 7D11 (ch7D11)

For construction of chimeric 7D11 IgG1, total RNA was extracted from7D11 hybridoma cells using a Qiagen RNeasy Mini Kit (Cat #74104) and themanufacturer's suggested protocol. Using the N-terminal amino acidsequences obtained for the light and heavy chains of the 7D11 monoclonalantibody, PCR primers specific for each chain were designed. Reverseprimers for RT-PCR were designed to match the framework 4 of the genefamily corresponding to the N-terminal sequence. Primers were alsodesigned to add desired restriction sites for cloning. For the lightchain these were EcoRV at the N-terminus, and KpnI at 3′ end ofFramework 4. For the heavy chain, sites added were BsiWI at theN-terminus, and ApaI slightly downstream of the VH-CH1 junction. Primersequences are as follows:

7D11LCF.EcoRV (7D11 light chain forward primer): (SEQ ID NO: 2)5′- GATCGATATCYTGCTSACMCARTGTCCAGC -3′ (B = G/T/C , K = G/T, Y =C/T, M = A/C, R = A/G, D = G/A/T. S = G/C, H = A/T/C)C7F7LCR.KpnI (7D11 light chain reverse primer): (SEQ ID NO: 3)5′- TTTDAKYTCCAGCTTGGTACC- 3′ (B = G/T/C , K = G/T, Y = C/T, M =A/C, R = A/G, D = G/A/T. S = G/C,  H = A/T/C)1D1(IIID-2)HCF.BsiWI (7D11 heavy chain forward primer): (SEQ ID NO: 4)5′- GATCGACGTACGCTGARGTGCARYTGGTGGARTCTGG- 3′ (B = G/T/C , K = G/T, Y =C/T, M = A/C, R = A/G, D = G/A/T. S = G/C,  H = A/T/C)C7F7HCR.ApaI (7D11 heavy chain reverse primer): (SEQ ID NO: 5)5′- ACAGTGGGCCCTTGGTGGAGGCTGMRGAGACDGTGASHRDRGT- 3′ (B = G/T/C , K =G/T, Y = C/T, M = A/C, R = A/G, D = G/A/T. S = G/C,  H = A/T/C)

RT-PCR reactions for light and heavy chains were carried out using aQiagen One-step RT-PCR kit (Cat #210210) and the suggested reactionmixes and conditions. pRK vectors for mammalian cell expression of IgGshave been previously described (Gorman et al., DNA Prot Eng Tech 2:3-10(1990). Vectors have been modified and have incorporated certainendonuclease restriction enzyme recognition sites to facilitate cloningand expression (Shields et al., J Biol Chem 2000; 276: 6591-6604).Amplified VL was cloned into a pRK mammalian cell expression vector(pRK.LPG3.HumanKappa) containing the human kappa constant domain.Amplified V11 was inserted to a pRK mammalian cell expression vector(pRK.LPG4.HumanHC) encoding the full-length human IgG1 constant domain.

The DNA sequence was obtained for the entire coding region of theresultant murine-human chimeric light (FIG. 1) and heavy (FIG. 3) chainsfor anti-human FcRH5 (ch7D11). The encoded polypeptide for themurine-human chimeric light and heavy chains encoded by the DNAs areshown in FIGS. 2 and 4, respectively. After DNA sequencing, theexpression of the plasmids were analyzed.

The plasmids were transiently transfected in 293 (anadenovirus-transformed human embryonic kidney cell line (Graham et al.,J. Gen. Virol., 36: 59-74 (1977)). Specifically, 293 cells were split onthe day prior to transfection, and plated in serum-containing medium. Onthe following day, double-stranded DNA prepared as a calcium phosphateprecipitate was added, followed by pAdVAntage™ DNA (Promega, Madison,Wis.), and cells were incubated overnight at 37° C. Cells were culturedin serum-free medium and harvested after 4 days. The antibody proteinswere purified from the transfected cell supernatants on proteinA columnsand then buffer exchanged into 10 mM sodium succinate, 140 mM NaCl, pH6.0, and concentrated using a Centricon-10 (Amicon). The identity of theproteins confirmed by N-terminal sequencing. Protein concentrations weredetermined by quantitative amino acid analysis. The antibodies weretested for binding to human FcRH5 by FACS in SVT2 cells as describedbelow.

2. Chimeric Anti-Human FcRH5 Antibody 10A8 (ch10A8)

Antibody 10A8 was derived as members of a panel of anti-human FcRH5hybridomas. Mouse/human chimeras of these antibodies were constructedusing RT-PCR to amplify the variable domains of hybridoma light andheavy chains using total mRNA as substrate. Light chain variable domainswere cloned into vector pRK.LPG3.HumanKappa containing the humanconstant kappa domain, while the heavy chain variable domains werecloned into vector pRK.LPG4.HumanHC encoding the full-length human IgG1constant domains. DNA sequencing allowed identification of framework andCDR regions of the variable domains. Chimeric and humanized versions of10A8 are described in U.S. Provisional Patent Application Nos.61/211,695 filed Apr. 1, 2009, 61/166,217 filed Apr. 2, 2009, and61/266,972 filed Dec. 4, 2009, which are each incorporated by referenceherein in their entirety.

3. Chimeric Anti-Human FcRH5 Antibody 13G9 (ch13G9)

Antibody 13G9 was derived as members of a panel of anti-human FcRH5hybridomas. Mouse/human chimeras of these antibodies were constructedusing RT-PCR, as described above, to amplify the variable domains ofhybridoma light and heavy chains using total mRNA as substrate. Lightchain variable domains were cloned into vector pRK.LPG3.HumanKappacontaining the human constant kappa domain, while the heavy chainvariable domains were cloned into vector pRK.LPG4.HumanHC encoding thefull-length human IgG1 constant domains. The DNA sequence was obtainedfor the entire coding region of the resultant murine-human chimericlight (FIG. 5) and heavy (FIG. 7) chains for anti-human FcRH5 (ch13G9),which allowed for identification of framework and CDR regions of thevariable domains. The encoded polypeptide for the 13G9 murine-humanchimeric light and heavy chains encoded by the DNAs are shown in FIGS. 6and 8, respectively.

Sequences of Primers Used for RT-PCR are as Follows:

13G9LCF.EcoRV (13G9 light chain forward primer): (SEQ ID NO: 6)5′- GGAGTACATTCAGATATCGTGATGACCCARTCTCAGAAAATCATG- 3′ (B = G/T/C , K =G/T, Y = C/T, M = A/C, R = A/G, D = G/A/T. S = G/C, H = A/T/C)CA1808 RsrII (13G9 light chain reverse primer): (SEQ ID NO: 7)5′- GGTGCAGCCACGGTCCGTTTGATTTCCAGCTTGGTGCCTCCACC- 3′MM102hc.f. PvuII (13G9 heavy chain forward primer): (SEQ ID NO: 8)5′- GGA GTA CAT TCA CAG ATC CAG CTG GTG CAG TCT GGA CCT GAG CTTAAG AAG- 3′ CA2172.ApaI (13G9 heavy chain reverse primer):(SEQ ID NO: 9) 5′-GACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGASTGWGGTTCC- 3′(B = G/T/C , K = G/T, Y = C/T, M = A/C, R = A/G, D = G/A/T. S = G/C, H =A/T/C)

D. Generation of Humanized Anti-Human FcRH5 Antibody 13G9

Residue numbers are according to Kabat (Kabat et al., Sequences ofproteins of immunological interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Single letteramino acid abbreviations are used. DNA degeneracies are representedusing the IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T,K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y=C/T).

1. Humanized Anti-FcRH5 Antibody Graft

Various humanized anti-FcRH5 antibodies were generated. The VL and VHdomains from murine FcRH5 antibody (13G9) were aligned with the humanconsensus VL kappa I (huKI) and human subgroup III consensus VH (huIII)domains. Alignment of the light chain variable domain of murine 13G9(mu13G9) with the human consensus sequence kappa I is shown in FIG. 9.The CDRs of 13G9 were engineered onto a mammalian expression vectorcontaining a mammalian signal sequence, the consensus sequence for thehuman V Kappa I variable domain, and a human constant kappa I domain.The CDRs were transferred using single oligonucleotides encoding eachCDR, and ssDNA of the acceptor plasmid, following the Kunkle mutagenesisprotocol. One murine residue, alanine 46, a Vernier residue in Framework2, was also transferred from the donor mAb to the acceptor plasmid.Residues grafted, and the resultant human 13G9 version one light chainvariable domain, are shown in FIG. 9.

Likewise, the CDRs of the heavy chain variable domain of murine 13G9were engineered onto a vector containing the mammalian signal sequence,the consensus sequence of human heavy subgroup III and sequencesencoding the full-length human IgG1 constant domains. Because there weredifferences noted in the murine vs human residues at several Vernierresidues known to influence conformation of the CDRs, murine frameworkresidues at positions 6, 69, 71, 73, 76 and 78 were also transferredfrom the donor to the acceptor plasmid. Alignments of the murine 13G9heavy variable domain, the human subgroup III variable domain, and theresulting human 13G9 version 1 (hu13G9.v1) heavy chain variable domainare shown in FIG. 10.

The chimera and version 1 of 13G9 were expressed in 293 cells andpurified as described above. Binding of resultant proteins was analyzedby FACS and Scatchard analysis (see below). Binding of version 1 of 13G9(hu13G9.v1) to cells expressing FcRH5 was essentially equivalent to thechimera, therefore, version 1 was carried forward for conjugationstudies.

It was noted that expression of humanized 13G9 was relatively low.Comparison of the framework sequences of murine 13G9 to several humansubgroup consensus sequences showed that 13G9 more closely resembledhuman subgroups VII and I than subgroup III. This suggested that eventhough high affinity binding to FcRH5 when the CDRs of 13G9 werehumanized to the framework of subgroup III, perhaps protein folding wasnot optimal. Therefore, framework changes were made to 13G9 version 1 toincrease homology to subgroup VII in order to improve expression.Alignments of the variable domains of the heavy chains of version 1,version 3 and version 8 are shown in FIG. 11. Substitutions made are asfollows: for version 3 (hu13G9.v3), V12K, V13K, V78A in the framework.In CDR H1, the substitution M34I was also made to improve packing. Forversion 8 (hu13G9.v8), substitutions were: L11V, V12K, V13K, L18V, R19K,L20V, K75V, V78A, N82I, N82aS, R83K, and M34I.

2. IgG Production

The plasmids were transiently transfected in 293 (anadenovirus-transformed human embryonic kidney cell line (Graham et al.,J. Gen. Virol., 36: 59-74 (1977)). Specifically, 293 cells were split onthe day prior to transfection, and plated in serum-containing medium. Onthe following day, double-stranded DNA prepared as a calcium phosphateprecipitate was added, followed by pAdVAntage™ DNA (Promega, Madison,Wis.), and cells were incubated overnight at 37° C. Cells were culturedin serum-free medium and harvested after 4 days. The antibody proteinswere purified from the transfected cell supernatants on proteinA columnsand then buffer exchanged into 10 mM sodium succinate, 140 mM NaCl, pH6.0, and concentrated using a Centricon-10 (Amicon). The identity of theproteins confirmed by N-terminal sequencing. Protein concentrations weredetermined by quantitative amino acid analysis. The antibodies weretested for binding to human FcRH5 by FACS in SVT2 cells as describedbelow.

3. Binding Analysis (FACS Analysis)

To determine the cross-reactivity of FcRH5 antibodies, binding of IgGvariants to SVT2 cells, expressing human FcRH1-6 were analyzed usingFACS analysis.

Briefly, approximately 10⁶ cells of SVT2 cells stably expressing gDtagged huFcRH1-6 in 100 μl were incubated with 1.0 ug of murine orchimeric Ab [Mu anti-gD (positive control), Mu anti-FcRH5 (1H4),(“mu1H4”) Mu anti-FcRH5 (4D10) (“mu4D10”), Mu anti-FcRH5 (6H1)(“mu6H1”), Mu anti-FcRH5 (7D11) (“mu7D11”), Ch anti-FcRH5 (7D11)(“ch7D11”), Mu anti-FcRH5 (8H6) (“mu8H6”), Mu anti-FcRH5 (9E2)(“mu9E2”), or Mu anti-FcRH5 (13G9) (“mu13G9”)]. Incubation of SVT2 cellsstably expressing the empty vector was incubated with the sameantibodies and used as a negative control. PE conjugated Ratanti-MuIgG₁, Rat anti-MuIgG_(2a+b), or Mu anti-HuIgG was used as thesecondary detection antibody. Results are shown in FIG. 15. All testedantibodies specifically bind FcRH5, expect for mu6H1, which cross-reactsto FcRH1. mu8H6 is a weak, but FcRH5 specific antibody, by FACSanalysis.

To further determine binding of the mouse FcRH5 antibodies, binding ofIgG variants to Raji cells, expressing human FcRH5 and 293T cellsexpressing cynomologous FcRH5, were analyzed using FACS analysis.

Briefly, approximately 10⁶ Raji cells stably transfected with humanFcRH5 and 293T cells transiently transfected with gD tagged cyno FcRH5were incubated 1.0 μg biotinylated mouse isotype control, biotinylatedmu anti-gD, biotinylated mu6H1, biotinylated mu13G9, or biotinylatedmu7D11 in 1000 FACS buffer (PBS, 0.5% BSA, 0.05% Sodium Azide). PEconjugated Streptavidin was used as the secondary detection reagent. Theresults are shown in FIGS. 16 and 17.

For additional FACS analysis of FcRH5 antibodies, 1.0 μg, 0.1 μg or 0.01μg of antibody was titrated per million cells of BJAB cells stablyexpressing huFcRH5 and SVT2 cells stably expressing cynoFcRH5. Briefly,approximately 10⁶ cells in 100 μl were incubated with varying amounts(1.0 ug, 01. ug or 0.01 ug) of murine Ab (‘mu1H4, mu4D10, mu6H1, mu7D11,mu9E2, mu10A8, mu140F5, or mu13G9) per million cells of BJAB cellsstably expressing huFcRH5 and SVT2 cells stably expressing cynoFcRH5.Incubation with 1.0 ug/10⁶ cells of the appropriate Mu IgG isotypecontrol was used as a negative control. PE conjugated Rat anti-MuIgG₁ orRat anti-MuIgG_(2a+b) was used as the secondary detection antibody.Results are shown in FIGS. 18 and 19. Mu anti-FcRH5 (1H4), Mu anti-FcRH5(6H1), Mu anti-FcRH5 (7D11), Ms anti-FcRH5 (10A8), Mu anti-FcRH5 (10F5),or Mu anti-FcRH5 (13G9) cross react with cynomolgus FcRH5 by FACSanalysis, and they can be used in preclinical safety studies assessingtarget dependent toxicity.

4. Affinity Determination (Scatchard Analysis)

To further determine binding of the FcRH5 murine antibodies and IgGclones, binding of iodinated IgG variants to OPM2 cells expressing humanFcRH5 and SVT2 cells expressing cynomologous FcRH5, Scatchard analysiswas performed (Table 9A).

For Scatchard analysis, 0.5 nM I¹²⁵ labeled murine or IgG cloneanti-FcRH5 was competed against unlabeled murine or IgG cloneanti-FcRH5, respectively, ranging from 50 to 0.02 nM (12 step 1:2 serialdilution) in the presence of a transfected OPM2 line stably expressinghuman-FcRH5 and a transfected SVT2 line stably expressingcynomologous-FcRH5. After a four hour incubation at 4° C., cells werewashed and cell pellet counts were read by a gamma counter (1470 WIZARDAutomatic Gamma Counter; Perkin Elmer, Walthem, Mass.). All points weredone in triplicate and counted for 10 minutes. The average CPM was usedfor Kd calculation using the New Ligand (Genentech, South San Francisco,Calif.) program. Results are shown in Table 9A.

TABLE 9A Affinity determination as determined by Scatchard analysis. KdSt. Cell Line PUR/Lot Ab (nM) Error BJAB.huFcRH5 48350-22 mu13G9 0.130.007 SVT2.cyFcRH5 48350-22 mu13G9 0.54 0.064 OPM2.huFcRH5 MM071207ch13G9 0.26 0.017 SVT2.cyFcRH5 MM071207 ch13G9 1.08 0.091 OPM2.huFcRH5PUR 16864 hu13G9.1a 0.20 0.012 (hu13G9.v1) SVT2.cyFcRH5 CA51434-50hu13G9.1a 0.56 0.027 (hu13G9.v1) BJAB.huFcRH5 50095-53 mu6H1 0.19 0.007SVT2.cyFcRHS 50095-93 mu6H1 0.38 0.027 OPM2.huFcRH5 CA51434-50hu13G9.1b* 0.30 0.027 SVT2.cyFcRHS CA51434-50 hu13G9.1b* 0.53 0.012*hu13G9.1b differs from hu13G9.1a, in that hu13G9.1b has a F instead ofa Y at position 87 of the light chain (see FIG. 9).

Example 2 Generation of Anti-FcRH5 Antibody Drug Conjugates (ADCs)

The drugs used for generation of antibody drug conjugates (ADCs) foranti-human FcRH5 included maytansinoid DM1 and DM4 and dolastatin 10derivatives monomethylauristatin E (MMAE) and monomethylauristatin F(MMAF). (See US20050276812, filed May 31, 2005 and US20050238649, filedNov. 5, 2004, both of which are incorporated by reference in theirentirety). MMAF, unlike MMAE, DM1 and DM4, is relative membraneimpermeable at neutral pH, so has relatively low activity as a freedrug, although it is very potent once in the cell. (Doronina et al.,Bioconjug. Chem., 17:114-123 (2006)). DM1, DM4, MMAE and MMAF aremitotic inhibitors that are at least 100 fold more cytotoxic than thevinca alkaloid mitotic inhibitors used in chemotherapeutic treatments ofNHLs (Doronina et al., Bioconjug. Chem., 17:114-123 (2006); Doronina etal., Nat. Biotechnol., 21: 778-784 (2003); Erickson et al., Cancer Res.,66: 4426-4433 (2006)). The linkers used for generation of the ADCs wereSMCC for DM1, SPDB for DM4 and MC or MC-vc-PAB for MMAE and MMAF. ForDM1 and DM4, the antibodies were linked to DM1 and DM4 through theε-amino group of lysine using the stable thioester linker SMCC(designated MCC after conjugation). Alternatively, for DM4, theantibodies were linked to DM4 through the e-amino group of lysine usingthe SPDB linker. DM1 attached via the SMCC linker is resistant tocleavage in reducing conditions. Further, SMCC-DM1 and SPDB-DM4 ADCsinduce cell toxicity if the ADC is internalized and targeted to thelysosome causing the release of lysine-N^(ε)-DM1, which is an effectiveanti-mitotic agent inside the cell, and when released from the cell,lysine-N^(ε)-DM1 is non-toxic (Erickson et al., Cancer Res., 66:4426-4433 (2006)) For MMAE and MMAF, the antibodies were linked to MMAEor MMAF through the cysteine by maleeimidocaproyl-valine-citruline(vc)-p-aminobenzyloxycarbonyl (MC-vc-PAB). For MMAF, the antibodies werealternatively linked to MMAF through the cysteine by maleeimidocaproyl(MC) linker. The MC-vc-PAB linker is cleavable by intercellularproteases such as cathepsin B and when cleaved, releases free drug(Doronina et al., Nat. Biotechnol., 21: 778-784 (2003)) while the MClinker is resistant to cleavage by intracellular proteases.

Antibody drug conjugates (ADCs) for anti-human FcRH5, using SMCC-DM1,were generated similar to the procedure described in U.S. applicationSer. No. 11/141,344, filed May 31, 2005, which is herein incorporated byreference in its entirety. Anti-human FcRH5 purified antibodies werebuffer-exchanged into a solution containing 50 mM potassium phosphateand 2 mM EDTA, pH 7.0. SMCC (Pierce Biotechnology, Rockford, Ill.) wasdissolved in dimethylacetamide (DMA) and added to the antibody solutionto make a final SMCC/Ab molar ratio of 10:1. The reaction was allowed toproceed for three hours at room temperature with mixing. TheSMCC-modified antibody was subsequently purified on a GE HealthcareHiTrap desalting column (G-25) equilibrated in 35 mM sodium citrate with150 mM NaCl and 2 mM EDTA, pH 6.0. DM1, dissolved in DMA, was added tothe SMCC antibody preparation to give a molar ratio of DM1 to antibodyof 10:1. The reaction was allowed to proceed for 4-20 hrs at roomtemperature with mixing. The DM1-modified antibody solution wasdiafiltered with 20 volumes of PBS to remove unreacted DM1, sterilefiltered, and stored at 4 degrees C. Typically, a 40-60% yield ofantibody was achieved through this process. The preparation wasusually >95% monomeric as assessed by gel filtration and laser lightscattering. Since DM1 has an absorption maximum at 252 nm, the amount ofdrug bound to the antibody could be determined by differentialabsorption measurements at 252 and 280 nm. Typically, the drug toantibody ratio was 3 to 4.

To generate anti-human FcRH5 SPDB DM4 antibody-drug conjugate with of3-4 drugs/Ab the antibody was first modified withSPDB[4-(2-Pyridyldithio)butyric acid-N-hydroxysuccinimide ester] togenerate dithiopyridyl groups. A 4-6 fold molar excess of SPDB toantibody is added and allowed to react for 90 min at RT. The reaction iscarried out in 50 mM potassium phosphate, pH 7 with 50 mM NaCl, 2 mMEDTA at an antibody concentration of 5-10 mgs/ml. Following the SPDBreaction, ethanol is added to a final concentration of 5% V/V and thethiol-containing DM4 drug is added at a 1.7 fold molar excess over SPDB.Incubation is carried out at room temperature for 16 hrs. The conjugateis purified by diafiltration, gel filtration, or cation exchangechromatography.

Antibody drug conjugates (ADC) for anti-human FcRH5, using MC-val-cit(vc)-PAB-MMAE drug linker were generated similar to the proceduredescribed in U.S. application Ser. No. 10/983,340, filed Nov. 5, 2004,which is herein incorporated by reference in its entirety. Purifiedanti-human FcRH5 antibody was dissolved in 500 mM sodium borate and 500mM sodium chloride at pH 8.0 and further treated with an excess of 100mM dithiothreitol (DTT). After incubation at 37 degrees C. for about 30minutes, the buffer is exchanged by elution over Sephadex G25 resin andeluted with PBS with 1 mM DTPA. The thiol/Ab value was checked bydetermining the reduced antibody concentration from the absorbance at280 nm of the solution and the thiol concentration by reaction with DTNB(Aldrich, Milwaukee, Wis.) and determination of the absorbance at 412nm. The reduced antibody dissolved in PBS was chilled on ice. The druglinker MC-val-cit (vc)-PAB-MMAE, in DMSO, was dissolved in acetonitrileand water, and added to the chilled reduced antibody in PBS. After anhour incubation, an excess of maleimide was added to quench the reactionand cap any unreacted antibody thiol groups. The reaction mixture wasconcentrated by centrifugal ultrafiltration and the antibody drugconjugate, was purified and desalted by elution through G25 resin inPBS, filtered through 0.2 μm filters under sterile conditions, andfrozen for storage.

Example 3 Preparation of Cysteine Engineered Anti-FcRH5 Antibodies

Preparation of cysteine engineered anti-FcRH5 antibodies was performedas disclosed herein. Cysteine residues may be added to the light chain,the heavy chain or the Fc region of an antibody to enable conjugation tovarious drugs. In the preparation of the cysteine engineered anti-FcRH5antibody 13G9, DNA encoding the light chain was mutagenized tosubstitute cysteine for valine at Kabat position 205 as shown in FIG.14. DNA encoding the heavy chain was mutagenized to substitute cysteinefor alanine at EU position 118 in the heavy chain (sequential position118, Kabat number 114) as shown in FIG. 13. The Fc region of anti-FcRH5antibodies may be mutagenized to substitute cysteine for serine at EUposition 400 in the heavy chain Fc region (sequential position 400;Kabat number 396).

Example 4 In Vivo Tumor Cell Killing Assay Using Murine Anti-FcRH5Antibody Drug Conjugates

A. Xenografts

To test the efficacy of various anti-FcRH5 antibodies, murine anti-humanantibodies were conjugated to DM1 and the effect of the conjugatedvariant on tumors in mice were analyzed.

Specifically, the ability of the antibodies to regress tumors inBJAB-luciferase cells (Burkitt's lymphoma cell line stably transfectedwith human FcRH5.

After transfection of pRK.CMV.PD.nbe.FcRH5 into BJAB-luciferase cellsand puromycin (Calbiochem, San Diego, Calif.) selection, surviving cellswere FACS autocloned for mid range expressers with anti-FcRH5 antibodies(7D11). Post autoclone, the BJAB-luciferase cell line expressing FcRH5most closely resembling plasma cell expression, as determined by FACS,was chosen by FACS analysis using anti-FcRH5 antibodies (7D11). As anegative control, BJAB-luciferase transfected, in the same manner, withthe empty vector pRK.CMV.PD.nbe were used.

For analysis of efficacy mouse antibodies, female CB17 ICR SCID mice(6-8 weeks of age from Charles Rivers Laboratories; Hollister, Calif.)were inoculated subcutaneously with 2×10⁷ BJAB-FcRH5 cells via injectioninto the flanks of CB17 ICR SCID mice and the xenograft tumors wereallowed to grow to an average of 128 mm³. Day 0 refers to the day thetumors were an average of 100-250 mm³ and when the first/or only dose oftreatment was administered, unless indicated specifically below. Tumorvolume was calculated based on two dimensions, measured using calipers,and was expressed in mm³ according to the formula: V=0.5a×b², where aand b are the long and the short diameters of the tumor, respectively.Data collected from each experimental group were expressed as mean±SE.Groups of 8 mice were treated with a single intravenous (i.v.) dose of400 μg of antibody-linked drug/m² mouse (corresponding to ˜8-10 mgADC/kg of mouse) with anti-FcRH5 ADC or control antibody-drugconjugates. Tumors were measured twice a week throughout the experiment.Body weights of mice were measured either once a week throughout theexperiment. Mice were euthanized before tumor volumes reached 2000 mm³or when tumors showed signs of impending ulceration. All animalprotocols were approved by an Institutional Animal Care and UseCommittee (IACUC).

Linkers between the antibody and the toxin that were used were thioethercrosslinker SMCC for DM1. Additional linkers may include disulfidelinker SPP or thioether crosslinker SMCC for DM1 or MC orMC-valine-citrulline(vc)-PAB or (a valine-citrulline (vc)) dipeptidelinker reagent) having a maleimide component and apara-aminobenzylcarbamoyl (PAB) self-immolative component formonomethylauristatin E (MMAE) or monomethylauristan F (MMAF). Toxinsused were DM1. Additional toxins may include MMAE or MMAF.

Antibodies for this experiment included mu7D11, described in Chang etal. PCT/US2004/043514 (WO 2005/063299), which is incorporated byreference herein in its entirety, as well as anti-FcRH5 described herein(see Example 1B, such as mu7D11, mu6H11, mu10A8, mu13G9 and mu9E2).

Negative controls included anti-gp-120 based conjugates (SMCC-DM1).

B. Results

1. BJAB-FcRH5 Xenografts

In a 48 day time course with drug conjugates and doses as shown mu7D11,mu6H1, mu10A8, mu13G9, and mu9E2 ADC, showed inhibition of tumor growthin SCID mice with BJAB-FcRH5 tumors compared to negative control, antigp-120 SMCC-DM1. ADCs were administered in two doses (as indicated belowin Table 10) at day 0 and day 7 for all ADCs and controls. Specifically,all of the anti-FcRH5 ADCs significantly inhibited tumor growth (FIG.20).

Further, in the same study, the percent body weight change in the first17 days was determined in each dosage group. The results (FIG. 21)indicated administration of these murine-anti-FcRH5 ADCs did not cause asignificant decrease in body weight or weight loss during this time.

Even further, in Table 10, the number of mice out of the total numbertested showing Partial Regression (PR; where the tumor volume at anytime after administration dropped below 50% of the tumor volume measuredat day 0), Complete Remission (CR; where the tumor volume at any timeafter administration dropped to 0 mm3) are indicated (NA=NotApplicable); (TI=number of observable tumors at end of study).

TABLE 10 Dose Drug - DM1 Dose Ab Drug ratio Antibody administered(Treatment) TI PR CR (μg/m²) (mg/kg) (Drug/Ab) Mu-anti-gp120 8/8 0 0 —10.8 — Mu-anti-gp120-mcc-dm1 8/8 0 0 400 8.4 3.2 Mu-anti-FcRH5 (7D11)8/8 0 0 — 10.8 — Mu-anti-FcRH5 (7D11)-mcc-dm1 8/8 0 0 400 9 3  Mu-anti-FcRH5 (6H1)-mcc-dm1 6/8 1 2 400 10.8 2.5 Mu-anti-FcRH5(10A8)-mcc-dm1 7/8 3 1 400 10 2.7 Mu-anti-FcRH5 (13G9)-mcc-dm1 7/8 0 1400 8.4 3.2 Mu-anti-FcRH5 (9E2)-mcc-dm1 8/8 2 0 400 10.4 2.6

Example 5 Assay for Inhibition of In Vivo Tumor Growth by MouseAnti-FcRH5 Mab Drug Conjugates

A. Xenografts

To test the efficacy of mu13G9 drug conjugates with -SPDB-DM4, SMCC-DM1,MC-vc-PAB-MMAE, and MC-MMAF, the effects of the mu13G9 drug conjugateson tumors in mice were analyzed.

Specifically, the ability of the antibodies to regress tumors in OPM2cells (Multiple myeloma cell line) stably transfected with human FcRH5was examined.

After transfection of linearized pRK.CMV.PD.nbe.FcRH5 into OPM2 cellsand puromycin (Calbiochem, San Diego, Calif.) selection, surviving cellswere separated for FcRH5 expressing cells with biotin (PierceBiotechnology, Rockford, Ill.) labeled anti-FcRH5.7D11 and MACSanti-Biotin microbeads on an MACS LS column (Miltenyi Biotec, Auburn,Calif.). Post separation FcRH5 expression was confirmed by FACS analysisusing anti-FcRH5 antibodies (10A8) and (13G9) and an OPM2 cell linetransfected, in the same manner, with the empty vector pRK.CMV.PD.nbe asa negative control.

For analysis of efficacy of mu13G9 drug conjugates with SPDB-DM4,SMCC-DM1, MC-vc-PAB-MMAE, and MC-MMAF, female CB17 ICR SCID mice (6-8weeks of age from Charles Rivers Laboratories; Hollister, Calif.) wereinoculated subcutaneously with 2×10⁷ OPM2-FcRH5 cells via injection intothe flanks of CB17 ICR SCID mice and the xenograft tumors were allowedto grow to an average of 215 mm³. Day 0 refers to the day the tumorswere an average of 100-250 mm³ and when the first/or only dose oftreatment was administered, unless indicated specifically below. Tumorvolume was calculated based on two dimensions, measured using calipers,and was expressed in mm³ according to the formula: V=0.5a×b², where aand b are the long and the short diameters of the tumor, respectively.Data collected from each experimental group were expressed as mean±SE.Groups of 8 mice were treated with a single intravenous (i.v.) dose of10 mg ADC/kg of with anti-FcRH5 ADC or control antibody-drug conjugates.Tumors were measured twice a week throughout the experiment. Bodyweights of mice were measured once a week throughout the experiment.Mice were euthanized before tumor volumes reached 3000 mm³ or whentumors showed signs of impending ulceration. All animal protocols wereapproved by an Institutional Animal Care and Use Committee (IACUC).

Negative controls included anti-gp-120 based conjugates (SPDB-DM4,SMCC-DM1, MC-vc-PAB-MMAE, and MC-MMAF).

B. Results

1. OPM2-FcRH5 Xenografts

In a 50 day time course with drug conjugates and doses as shown in Table11, mu13G9 SPDB-DM4, SMCC-DM1, MC-vc-PAB-MMAE, and MC-MMAF ADCs, showedinhibition of tumor growth in SCID mice with OPM-FcRH5 tumors comparedto negative control, anti gp-120 SPDB-DM4, SMCC-DM1, MC-vc-PAB-MMAE, andMC-MMAF ADC. ADCs were administered in a two doses at day 0 and day 7for all ADCs and controls. Specifically, all of the anti-FcRH5 ADCssignificantly inhibited tumor growth (FIG. 22).

Further, in the same study, the percent body weight change in the first14 days was determined in each dosage group. The results (FIG. 23)indicated administration of these murine anti-FcRH5 ADCs did not cause asignificant decrease in body weight or weight loss during this time.

Even further, in Table 11, the number of mice out of the total numbertested showing Partial Regression (PR; where the tumor volume at anytime after administration dropped below 50% of the tumor volume measuredat day 0), Complete Remission (CR; where the tumor volume at any timeafter administration dropped to 0 mm3) are indicated (NA=NotApplicable); (TI=number of observable tumors at end of study).

TABLE 11 Dose Drug - DM1 Dose Ab Drug ratio Antibody administered(Treatment) TI PR CR (μg/m²) (mg/kg) (Drug/Ab) Vehicle* 7/7 0 0 n/a —n/a Mu-anti-gp120-spdb-DM4 7/7 0 0 329 10 3.0 Mu-anti-gp120-smcc-dml 7/70 0 332 10 3.2 Mu-anti-gp120-MC-MMAF 7/7 0 0 371 10 3.6Mu-anti-gp120-MCvcPAB-MMAE 7/7 0 0 293 10 2.9 Mu-anti-FcRH5(13G9)-spdb-DM4 5/6 5 1 362 10 3.3 Mu-anti-FcRH5 (13G9)-smcc-dml 7/7 0 0363 10 3.5 Mu-anti-FcRH5 (13G9)-MC-MMAF 7/7 0 0 360 10 3.5 Mu-anti-FcRH5(13G9)-MCvcPAB-MMAE 7/7 0 0 363 10 3.6 *Vehicle = 20 mM histidineacetate, pH 5.5, 240 mM sucrose, 0.02% PS20

Example 6 Assay for In Vitro Cell Proliferation Reduction by Anti-FcRH5Mab Drug Conjugates

The in vitro potency of mu13G9 drug conjugates (including mu anti-FcRH5(13G9)-Naked, mu anti-FcRH5 (13G9)-SMCC-DM1, mu anti-FcRH5(13G9)-SPDB-DM4, mu anti-FcRH5 (13G9)-MC-vc-PAB-MMAE, and mu anti-FcRH5(13G9)-MC-MMAF) was measured by a cell proliferation assay withtransfected OPM2 line stably expressing human-FcRH5(OPM2.CMV.PD.FcRH5.5P.2). The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670). This cell proliferation assay determines thenumber of viable cells in culture based on quantitation of the ATPpresent, an indicator of metabolically active cells (Crouch et al., J.Immunol. Metho., 160: 81-88 (1993); U.S. Pat. No. 6,602,677). TheCellTiter-Glo® Assay was conducted in 96 well format, making it amenableto automated high-throughput screening (HTS) (Cree et al., AntiCancerDrugs, 6:398-404 (1995)). The homogeneous assay procedure involvesadding the single reagent (The CellTiter-Glo® Reagent) directly to cellscultured in serum-supplemented medium.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The substrate, Beetle Luciferin, is oxidatively decarboxylatedby recombinant firefly luciferase with concomitant conversion of ATP toAMP and generation of photons. Viable cells are reflected in relativeluminescence units (RLU). Data can be recorded by luminometer or CCDcamera imaging device. The luminescence output is presented as RLU,measured over time. % RLU is normalized RLU percentage compared to a“non-drug-conjugate” control. Alternatively, photons from luminescencecan be counted in a scintillation counter in the presence of ascintillant. The light units can be represented then as CPS (counts persecond).

Efficacy of humanized antibody-drug conjugates were measured by a cellproliferation assay employing the following protocol, adapted fromCellTiter Glo Luminescent Cell Viability Assay, Promega Corp. Technicalbulletin TB288; Mendoza et al., Cancer Res., 62: 5485-5488 (2002)):

1. An aliquot of 50 μl of cell culture containing about 75,000OPM2.CMV.PD.FcRH5.SP.2 cells in medium was deposited in each well of a96-well, opaque-walled plate.

2. mu13G9 Ab or ADC (50 ul) was added to triplicate experimental wellsto final concentration of 10000, 3333, 1111, 370, 123, 41, 14, 4.6 or1.5 ng/mL, with “non-drug conjugate” control wells receiving mediumalone, and incubated for 3 days.

3. The plates were equilibrated to room temperature for approximately 30minutes.

4. CellTiter-Glo Reagent (100 μl) was added.

5. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

6. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

7. Luminescence was recorded and reported in graphs as % RLU (relativeluminescence units).

8. As a control, the same cell line was run in parallel with irrelevantnon-binding mouse anti-GP120 Abs or ADCs (including mu anti-GP120 Naked,mu anti-GP120-SMCC-DM1, mu anti-GP120-SPDB-DM4, muanti-GP120-MC-vc-PAB-MMAE, and mu anti-GP120-MC-MMAF).

Media: OPM2.CMV.PS.FcRH5.SP.2 cells grow in RPMI1640/20% FBS/2 mMglutamine/1.0 ug/ml Puromycin.

Efficacy of mouse antibody-drug conjugates is shown in FIGS. 24-25.Results of this assay are stated in mg/ml as the IC50. The IC50 beingthe concentration of the antibody or ADC required for a 50% inhibitionin cell growth.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

Example 7 Assay for Inhibition of In Vivo Tumor Growth by HumanizedAnti-FcRH5 Drug Conjugates and Velcade®

A. Xenografts

To test the efficacy of Humanized (13G9) drug conjugate withMC-vc-PAB-MMAE) in combination with Velcade® (bortezomib), the effectsof the conjugated antibody, alone and in combination with Velcade®, ontumors (multiple myeloma OPM2-FcRH5 xenograft) in mice are analyzed.

Specifically, the ability of the antibody and Velcade® to regress tumorsin OPM2 cells (Multiple myeloma cell line) stably transfected with humanFcRH5 is examined. After transfection of linearized pRK.CMV.PD.nbe.FcRH5into OPM2 cells and puromycin (Calbiochem, San Diego, Calif.) selection,surviving cells are separated for FcRH5 expressing cells with biotin(Pierce Biotechnology, Rockford, Ill.) labeled anti-FcRH5.7D11 and MACSanti-Biotin microbeads on an MACS LS column (Miltenyi Biotec, Auburn,Calif.). Post separation FcRH5 expression is confirmed by FACS analysisusing anti-FcRH5 antibodies (13G9) and an OPM2 cell line transfected, inthe same manner, with the empty vector pRK.CMV.PD.nbe as a negativecontrol.

For analysis of efficacy of hu anti-FcRH5 13G9 conjugated withMC-vc-PAB-MMAE alone and in combination with Velcade®, female CB17 ICRSCID mice (6-8 weeks of age from Charles Rivers Laboratories; Hollister,Calif.) are inoculated subcutaneously with 2×10⁷ OPM2-FcRH5 cells viainjection into the flanks of CB17 ICR SCID mice and the xenograft tumorsare allowed to grow to an average of 331 mm³. Day 0 refers to the daythe tumors are 172-511 mm³ and when the first/or only dose of treatmentis administered, unless otherwise indicated. Tumor volume is calculatedbased on two dimensions, measured using calipers, and is expressed inmm3 according to the formula: V=0.5a×b², where a and b are the long andthe short diameters of the tumor, respectively. Data collected from eachexperimental group are expressed as mean±SE. Mice are randomized intonine groups of about 9 mice each and dosed. Group 1 receives a singleintravenous (IV) injection of histidine buffer on Day 0 plus IVinjections of 0.9% sodium chloride (saline) twice weekly for two weekson Days 0, 3, 7, and 10 as a vehicle control. Group 2 receives a singleIV injection of 10 mg/kg Hu anti-Her2 Trastuzumab-MC-vc-PAB-MMAE, CNJ1135, on Day 0 as a conjugate control. Group 3 receives IV injections of1 mg/kg Velcade® twice weekly for two weeks on Days 0, 3, 7, and 10.Groups 4-5 receive a single IV injection of 4 or 10 mg/kg thio huanti-FcRH5(13G9)-HC-MC-vc-PAB-MMAE, CNJ 1465, respectively, on Day 0.Group 6-7 receives a single IV injection of 4 or 10 mg/kg huanti-FcRH5(13G9)-MC-vc-PAB-MMAE, CNJ 1575, respectively, on Day 0. Group8 receives a single IV injection of 4 mg/kg huanti-FcRH5(13G9)-MC-vc-PAB-MMAE, CNJ 1575, on Day 0 plus IV injectionsof 1 mg/kg Velcade® twice weekly for two weeks on Days 0, 3, 7, and 10.Tumors are measured twice a week throughout the experiment. Body weightsof mice are measured twice a week for the first three weeks. Mice areeuthanized before tumor volumes reached 3000 mm³ or when tumors showedsigns of impending ulceration. All animal protocols are approved by anInstitutional Animal Care and Use Committee (IACUC).

Negative control includes anti-HER2 (HERCEPTIN®) (trastuzumab)) basedconjugate (MC-vc-PAB-MMAE).

B. Results

1. OPM2-FcRH5 Xenografts

At the end of the study with the drug conjugate alone and in combinationwith Velcade®, hu13G9 conjugated with -MC-vc-PAB-MMAE combined withVelcade® is assayed for inhibition of tumor growth in SCID mice withOPM2-FcRH5 tumors compared to negative control, humanized anti-Her2conjugated with -MC-vc-PAB-MMAE (Hu anti-Her2Trastuzumab-MC-vc-PAB-MMAE). Further, any percent body weight changes inthe first 24 days are determined in each dosage group. The number ofmice out of the total number tested showing Partial Regression (PR;where the tumor volume at any time after administration dropped below50% of the tumor volume measured at day 0), Complete Remission (CR;where the tumor volume at any time after administration dropped to 0mm³) are also determined (NA=Not Applicable); (TI=number of observabletumors at end of study).

Example 9 Assay for Inhibition of In Vivo Tumor Growth by HumanizedAnti-FcRH5 Drug Conjugates

A. Xenografts

To test the efficacy of Humanized (13G9) drug conjugate with —SPDB-DM4in combination with Velcade®, the effects of the conjugated antibody,alone and in combination with Velcade®, on tumors (multiple myelomaOPM2-FcRH5 xenograft) in mice are analyzed.

Specifically, the ability of the antibody and Velcade® to regress tumorsin OPM2 cells (Multiple myeloma cell line) stably transfected with humanFcRH5 is examined. After transfection of linearized pRK.CMV.PD.nbe.FcRH5into OPM2 cells and puromycin (Calbiochem, San Diego, Calif.) selection,surviving cells are separated for FcRH5 expressing cells with biotin(Pierce Biotechnology, Rockford, Ill.) labeled anti-FcRH5.7D11 and MACSanti-Biotin microbeads on an MACS LS column (Miltenyi Biotec, Auburn,Calif.). Post separation FcRH5 expression is confirmed by FACS analysisusing anti-FcRH5 antibodies (13G9) and an OPM2 cell line transfected, inthe same manner, with the empty vector pRK.CMV.PD.nbe as a negativecontrol.

For analysis of efficacy of Hu anti-FcRH5 13G9 conjugated to -SPDB-DM4alone and in combination with Velcade®, female CB17 ICR SCID mice (6-8weeks of age from Charles Rivers Laboratories; Hollister, Calif.) areinoculated subcutaneously with 2×10⁷ OPM2-FcRH5 cells via injection intothe flanks of CB17 ICR SCID mice and the xenograft tumors are allowed togrow to an average of 306 mm³. Day 0 refers to the day the tumors were197-499 mm³ and when the first/or only dose of treatment isadministered, unless otherwise indicated. Tumor volume is calculatedbased on two dimensions, measured using calipers, and is expressed inmm3 according to the formula: V=0.5a×b², where a and b are the long andthe short diameters of the tumor, respectively. Data collected from eachexperimental group are expressed as mean±SE. Mice are randomized intosix groups of about 9 mice each and dosed. Group 1 receives intravenous(IV) injections of 1 mg/kg Velcade® twice weekly for two weeks on Days0, 3, 7, and 10. Group 2 receives a single IV injection of histidinebuffer on Day 0 plus IV injections of 0.9% sodium chloride (saline)twice weekly for two weeks on Days 0, 3, 7, and 10 as a vehicle control.Group 3 receives a single IV injection of 4 mg/kg Hu anti-Her2Trastuzumab-SPDB-DM4, CNJ 1433, on Day 0 as a conjugate control. Group 4receives a single IV injection of 4 mg/kg Hu anti-FcRH5 13G9 SPDB-DM4,CNJ 1503, on Day 0. Group 5 receives a single IV injection of 4 mg/kg Huanti-Her2 Trastuzumab-SPDB-DM4, CNJ 1433, on Day 0 plus IV injections of1 mg/kg Velcade® twice weekly for two weeks on Days 0, 3, 7, and 10.Group 6 receives a single IV injection of 4 mg/kg Hu anti-FcRH5 13G9SPDB-DM4, CNJ 1503, on Day 0 plus IV injections of 1 mg/kg Velcade®twice weekly for two weeks on Days 0, 3, 7, and 10. Tumors are measuredtwice a week throughout the experiment. Body weights of mice aremeasured twice a week for the first three weeks. Mice are euthanizedbefore tumor volumes reached 3000 mm³ or when tumors showed signs ofimpending ulceration. All animal protocols are approved by anInstitutional Animal Care and Use Committee (IACUC).

Negative control includes anti-HER2 (HERCEPTIN®) (trastuzumab)) basedconjugate (SPDB-DM4).

B. Results

1. OPM2-FcRH5 Xenografts

At the end of the study with the drug conjugate alone and in combinationwith Velcade®, humanized anti-FcRH5 (13G9) conjugated with -SPDB-DM4combined with Velcade® is assayed for inhibition of tumor growth in SCIDmice with OPM2-FcRH5 tumors compared to negative control, humanizedanti-Her2 conjugated with -SPDB-DM4 (Hu anti-Her2 Trastuzumab-SPDB-DM4).Further, the percent body weight change in the first 21 days isdetermined in each dosage group. The number of mice out of the totalnumber tested showing Partial Regression (PR; where the tumor volume atany time after administration dropped below 50% of the tumor volumemeasured at day 0), Complete Remission (CR; where the tumor volume atany time after administration dropped to 0 mm³) are also determined(NA=Not Applicable); (TI=number of observable tumors at end of study).

Example 10 Assay for Inhibition of In Vivo Tumor Growth by HumanizedAnti-FcRH5 Drug Conjugates

A. Xenografts

To test the efficacy of Humanized (13G9) drug conjugate with-MC-vc-PAB-MMAE) in combination with Revlimid® and Revlimid® plusDexamethasone, the effects of the conjugated antibody in combinationwith Revlimid® and Revlimid® plus Dexamethasone, on tumors (multiplemyeloma OPM2-FcRH5 xenograft) in mice are analyzed.

Specifically, the ability of the antibody in combination with Revlimid®and Revlimid® plus Dexamethasone to regress tumors in OPM2 cells(Multiple myeloma cell line) stably transfected with human FcRH5 areexamined. After transfection of linearized pRK.CMV.PD.nbe.FcRH5 intoOPM2 cells and puromycin (Calbiochem, San Diego, Calif.) selection,surviving cells are separated for FcRH5 expressing cells with biotin(Pierce Biotechnology, Rockford, Ill.) labeled anti-FcRH5.7D11 and MACSanti-Biotin microbeads on an MACS LS column (Miltenyi Biotec, Auburn,Calif.). Post separation FcRH5 expression is confirmed by FACS analysisusing anti-FcRH5 antibodies (13G9) and an OPM2 cell line transfected, inthe same manner, with the empty vector pRK.CMV.PD.nbe as a negativecontrol.

For analysis of efficacy of Hu anti-FcRH5 13G9 conjugated to-MC-vc-PAB-MMAE in combination with Revlimid® and Revlimid® plusDexamethasone, female CB17 ICR SCID mice (6-8 weeks of age from CharlesRivers Laboratories; Hollister, Calif.) are inoculated subcutaneouslywith 2×10⁷ OPM2-FcRH5 cells via injection into the flanks of CB 17 ICRSCID mice and the xenograft tumors are allowed to grow to an average of446 mm³. Day 0 refers to the day the tumors are 263-630 mm³ and when thefirst/or only dose of treatment is administered, unless otherwiseindicated. Tumor volume is calculated based on two dimensions, measuredusing calipers, and is expressed in mm3 according to the formula:V=0.5a×b², where a and b are the long and the short diameters of thetumor, respectively. Data collected from each experimental group areexpressed as mean±SE. Mice are randomized into nine groups of about 8mice each and dosed. Group 1 receives daily intraperitoneal (IP)injections of a mixture of DMSO and MCT buffer on Days 0-13 as a vehiclecontrol. Group 2 receives a single intravenous (IV) injection of 6 mg/kgHu anti-Her2 Trastuzumab-MC-vc-PAB-MMAE, CNJ 1135, on Day 0 as aconjugate control. Group 3 receives a single IV injection of 6 mg/kg Huanti-FcRH5 13G9-MC-vc-PAB-MMAE, CNJ 1672, on Day 0 as a conjugatecontrol. Group 4 receives daily IP injections of 50 mg/kg Revlimid® onDays 0-13. Group 5 receives daily oral (PO) dosing of 3 mg/kgDexamethasone for four days followed by three days off for two cycles onDays 0-3 and 7-10. Group 6 receives daily IP injections of 50 mg/kgRevlimid® on Days 0-13 plus daily PO dosing of 3 mg/kg Dexamethasone forfour days followed by three days off for two cycles on Days 0-3 and7-10. Group 7 receives a single IV injection of 6 mg/kg Hu anti-Her2Trastuzumab-MC-vc-PAB-MMAE, CNJ 1135, on Day 0 plus daily IP injectionsof 50 mg/kg Revlimid® on Days 0-13 and daily PO dosing of 3 mg/kgDexamethasone for four days followed by three days off for two cycles onDays 0-3 and 7-10. Group 8 receives a single IV injection of 6 mg/kg Huanti-FcRH5 13G9-MC-vc-PAB-MMAE, CNJ 1672, on Day 0 plus daily IPinjections of 50 mg/kg Revlimid® on Days 0-13 and daily PO dosing of 3mg/kg Dexamethasone for four days followed by three days off for twocycles on Days 0-3 and 7-10. Group 9 receives a single IV injection of 6mg/kg Hu anti-FcRH5 13G9-MC-vc-PAB-MMAE, CNJ 1672, on Day 0 plus dailyIP injections of 50 mg/kg Revlimid® on Days 0-13. Tumors are measuredtwice a week throughout the experiment. Body weights of mice aremeasured twice a week throughout the experiment. If mice experience abody weight loss of greater than 15% compared to their body weight onDay 0, mice are weighed daily until weight regained or until body weightloss reaches greater than 20%. Mice are euthanized when they reach abody weight loss of greater than 20% compared to their body weight onDay 0, before tumor volumes reached 3000 mm³, or when tumors showedsigns of impending ulceration. All animal protocols are approved by anInstitutional Animal Care and Use Committee (IACUC).

Negative control includes anti-HER2 (HERCEPTIN®) (trastuzumab) basedconjugate (MC-vc-PAB-MMAE).

B. Results

1. OPM2-FcRH5 Xenografts

At the end of the study with the drug conjugate in combination withRevlimid® and Revlimid® plus Dexamethasone, humanized anti-FcRH5 (13G9)conjugated with -MC-vc-PAB-MMAE combined with Revlimid® and Revlimid®plus Dexamethasone is assayed for inhibition of tumor growth in SCIDmice with OPM2-FcRH5 tumors compared to negative control, humanizedanti-HER2 conjugated with -MC-vc-PAB-MMAE (Hu anti-Her2Trastuzumab-MC-vc-PAB-MMAE). Further, the percent body weight change inthe first 17 days is determined in each dosage group. The number of miceout of the total number tested showing Partial Regression (PR; where thetumor volume at any time after administration dropped below 50% of thetumor volume measured at day 0), Complete Remission (CR; where the tumorvolume at any time after administration dropped to 0 mm³) are alsodetermined (NA=Not Applicable); (TI=number of observable tumors at endof study).

Example 11 Production and Characterization of FcRH5 BispecificAntibodies

Construction and Production of Anti-CD3/FcRH5 Bispecific Antibodies.

The construction of an FcRH5 bispecific antibody, specifically ananti-CD3/FcRH5 protruberance-into-cavity bispecific antibody isdescribed below.

Various protruberance-into-cavity bispecific antibodies have beendescribed previously. See, e.g., U.S. Pat. Nos. 5,731,168 and 7,183,076;Ridgway et al., Prot. Eng. 9:617-621 (1996); Atwell et al., J. Mol.Biol. 270:26-35 (1997); Merchant et al., Nat. Biotechn. 16:677-681(1998). In certain instances, the C_(H)3 interface between the humanizedanti-CD3/CD4-IgG chimera previously described by Chamow et al. J.Immunol. 153:4268 (1994) was engineered to maximize the percentage ofheteromultimers that could be recovered from mammalian cell linescotransfected with expression constructs. As used herein,“heteromultimers” refers to a molecule, e.g., a bispecific antibody,comprising a least a first polypeptide and a second polypeptide, whereinthe second polypeptide differs in amino acid sequence from the firstpolypeptide by at least one amino acid residue.

As described in the documents cited in the preceding paragraph, thestrategy used to drive heteromultimer formation relied on introducingone or more “protruberance” mutations into a first polypeptide, e.g.,the C_(H)3 domain of one antibody H chain, and also introducing one ormore corresponding “cavity” mutations into a second polypeptide, e.g.,the C_(H)3 domain of a second antibody H chain. A “protuberance”mutation replaced a small amino acid with a larger one and a “cavity”mutation replaced a large amino acid with a smaller one. Furthermore, inaddition to the protruberance and cavity mutations, freethiol-containing residues were introduced at the interface of twopolypeptides, e.g., two C_(H)3 domains, which were shown to enhanceformation of heteromultimers. Various exemplaryprotruberance-into-cavity mutations and free thiol-containing residuesintroduced at polypeptide interfaces have been described. See, e.g.,U.S. Pat. Nos. 5,731,168 and 7,183,076; Ridgway et al., Prot. Eng.9:617-621 (1996); Atwell et al., J. Mol. Biol. 270:26-35 (1997);Merchant et al., Nat. Biotechn. 16:677-681 (1998).

The following steps are carried out to produce an anti-CD3/FcRH5bispecific antibody. E. coli expression plasmids encoding humanizedanti-CD3 light (L) and heavy (H) chain variants of the murine anti-CD3monoclonal Ab UCHT1 (U.S. Pat. Nos. 5,821,337 and 6,407,213; Shalaby etal., J. Exp. Med. 175: 217 and Rodrigues et al., Int. J. Cancer (Suppl.)7: 45 [1992]) are constructed. Many suitable E. coli expression plasmidsare known in the art, including pAK19. Carter et al., Bio/Tehcnology10:163-167 (1992). A mutation introducing either a protruberance or acavity into the C_(H)3 domain (as described above) of the humanizedanti-CD3 H chain is made, for example, by site-directed mutagenesis.Various site-directed mutagenesis methods are well known in the art.See, e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987). Inaddition, E. coli expression plasmids encoding humanized anti-FcRH5light and heavy chains, such humanized anti-FcRH5 light and heavy chainsas described herein, are constructed. A mutation introducing either acorresponding cavity (if the anti-CD3 C_(H)3 domain has a protruberancemutation) or a corresponding protruberance (if the anti-CD3 C_(H)3domain has a cavity mutation) in the C_(H)3 domain (as described above)of the humanized anti-FcRH5 H chain is made, for example, bysite-directed mutagenesis. Bispecific anti-CD3/FcRH5 antibodies areproduced by transforming the E. coli expression plasmids described aboveinto an appropriate E. coli strain, e.g., 33B6, using methods well knownin the art. See, e.g., Rodrigues et al., Cancer Res. 55:63-70 (1995).Antibody is secreted by the transformed E. coli grown in a 10 Lfermentor as described previously. Carter et al., Bio/Technology10:163-167 (1992). The antibodies are purified and analyzed usingprocedures known in the art. See, e.g., Atwell et al., J. Mol. Biol.270:26-35 (1997).

Cell Binding Assay:

To assess the ability of anti-CD3/FcRH5 bispecific antibodies to bind Bcells and T cells, human peripheral white blood cells are isolated fromwhole blood from healthy donors using a Ficoll gradient according tostandard procedures known in the art. About 1 million cells areincubated with anti-CD3/FcRH5 bispecific antibody on ice in buffercontaining 0.5% BSA and 2 mM EDTA in PBS. The cells are then washed andstained with FITC-conjugated goat (Fab′)₂ anti-human IgG, together withanti-CD8-APC and anti-CD138-PE and anti-CD38-PerCP-Cy5 antibodiesfollowing the manufacturer's protocol (BD Biosciences, San Diego,Calif.). Binding activities are assessed with a FACS-based assay usingFACSCalibur™ Flow Cytometer (BD Biosciences, San Diego, Calif.) andanalysis is carried out using Flowjo software (Tree Star, Ashland,Oreg.).

In Vitro Cell Killing Assay:

EJM-FcRH5.LSP.2 multiple myeloma cells (EJM cells, DSMZ ACC-560, stablytransfected with human FcRH5) are used as target cells and areco-cultured with total human peripheral white blood cells or CD8+ Tcells. If CD8+ T cells are used, they are purified from human PBMC bynegative sorting using a CD8+ T cell isolation kit, (Miltenyi Biotech,Auburn, Calif.) in 96-well round-bottom plates. 20,000 EJM-FcRH5.LSP.2cells are added per well with a ratio of target: effector cells=1:10,with or without anti-CD3/FcRH5 bispecific antibody. After approximately19 hours, cells are stained with labeled anti-CD38-FITC andanti-CD138-PE following the manufacturer's protocol (BD Biosciences, SanDiego, Calif.) and mixed with Fluoresbrite® Calibration Grade 6.0 MicronYG Microspheres (Polysciences, Inc., Warrington, Pa.). FACS analysis iscarried out by gating forward/sideward scatters and CD38-CD138 staining.Dead cells are excluded using propidium iodide staining according tostandard procedures. Specific killing activity of the anti-CD3/FcRH5bispecific antibody is calculated according to the following equation: %specific killing=(1-number of live EJM-FCRH5.LSP.2 cells withtreatment/number of live EJM-FCRH5.LSP.2 cells in effector &EJM-FCRH5.LSP.2 without anti-CD3/FcRH5 bispecific antibody)×100.

In Vivo Efficacy:

EJM-FcRH5.LSP.2 multiple myeloma cells are mixed with peripheral bloodmononuclear cells and injected subcutaneously into the right thoracicflank of female NOD.CD17-Prkdc^(scid)/J mice (Charles RiversLaboratories, Wilmington, Mass.). The inoculated mice are then randomlyseparated into control and treatment groups. Control groups areadministered vehicle or anti-CD3/Her2 control bispecific antibody.Treatment groups are administered anti-CD3/FcRH5 bispecific antibody.Bispecific antibodies are administered over a range of 0.01 to 10 mg/kg.Control or treatment is administered intravenously within 1-2 hours postcell inoculation and repeated daily for 5 consecutive days. Tumors aremeasured in two dimensions (length and width) using calipers twice atweek. Mice are clinically monitored twice a week. At the end of theexperiment, tumor size in the control-treated animals is compared totumor size in the anti-CD3/FcRH5 bispecific antibody-treated animals toassess the efficacy of the anti-CD3/FcRH5 bispecific antibody treatment.

Example 12 Assay for Inhibition of In Vivo Tumor Growth by HumanizedAnti-FcRH5 Drug Coniuuates

A. Xenografts

To test the efficacy of hu13G9 drug conjugates with MC-vc-PAB-MMAE atdifferent dosages, the effects of the conjugated antibody on tumors inmice are analyzed.

Specifically, the ability of the antibodies to regress tumors in EJMcells (Multiple myeloma cell line) stably transfected with human FcRH5are examined.

EJM (ACC-560) cells are a commercially available human multiple myelomacell line obtained from DSMZ (Germany). As EJM cells do not endogenouslyexpress FcRH5, the EJM cells were stably transfected with human FcRH5,resulting in the EJM.CMV.PD.FcRH5.LSP.2 cell line.

For analysis of efficacy of hu13G9 drug conjugates with MC-vc-PAB-MMAE,female CB17 ICR SCID mice (6-8 weeks of age from Charles RiversLaboratories; Hollister, Calif.) are inoculated subcutaneously with2×10⁷EJM2-FcRH5.LSP.2 cells via injection into the flanks of CB17 ICRSCID mice and the xenograft tumors are allowed to grow to an average of134 mm³. Day 0 refers to the day the tumors were an average of 100-250mm³ and when the first/or only dose of treatment is administered, unlessotherwise indicated. Tumor volume is calculated based on two dimensions,measured using calipers, and is expressed in mm³ according to theformula: V=0.5a×b², where a and b are the long and the short diametersof the tumor, respectively. Data collected from each experimental groupare expressed as mean±SE. Groups of mice are treated with a singleintravenous (i.v.) dose ranging from about 1 to about 8 mg ADC/kg ofwith anti-FcRH5 ADC or control antibody-drug conjugates (ADCs). Tumorsare measured twice a week throughout the experiment. Body weights ofmice are measured once a week for 2 weeks. Mice are euthanized beforetumor volumes reached 3000 mm³ or when tumors show signs of impendingulceration. Negative control include anti-HER2 (HERCEPTIN®(trastuzumab)) based conjugate (MC-vc-PAB-MMAE).

B. Results

1. EJM-FcRH5.LSP.2 Xenografts

At the end of the experiment, tumor size in the control-treated animalsis compared to tumor size in the anti-FcRH5 ADC-treated animals toassess the efficacy of the anti-FcRH5 ADC treatment. Specifically, thenumber of mice out of the total number tested showing Partial Regression(PR; where the tumor volume at any time after administration droppedbelow 50% of the tumor volume measured at day 0), Complete Remission(CR; where the tumor volume at any time after administration dropped to0 mm3) are also determined (NA=Not Applicable); (TI=number of observabletumors at end of study).

Example 13 Assay for Inhibition of In Vivo Tumor Growth by HumanizedAnti-FcRH5 Drug Conjugates

A. Xenografts

To test the efficacy of hu13G9 drug conjugates with SPDB-DM4 atdifferent dosages, the effects of the conjugated antibody on tumors inmice are analyzed.

Specifically, the ability of the antibodies to regress tumors in EJMcells (Multiple myeloma cell line) stably transfected with human FcRH5are examined.

EJM (ACC-560) cells are a commercially available human multiple myelomacell line obtained from DSMZ (Germany). As EJM cells do not endogenouslyexpress FcRH5, the EJM cells were stably transfected with human FcRH5,resulting in the EJM.CMV.PD.FcRH5.LSP.2 cell line.

For analysis of efficacy of hu13G9 drug conjugates with SPDB-DM4, femaleCB17 ICR SCID mice (6-8 weeks of age from Charles Rivers Laboratories;Hollister, Calif.) are inoculated subcutaneously with 2×10⁷EJM2-FcRH5.LSP.2 cells via injection into the flanks of CB17 ICR SCIDmice and the xenograft tumors are allowed to grow to an average of 134mm³. Day 0 refers to the day the tumors are an average of 100-250 mm³and when the first/or only dose of treatment is administered, unlessotherwise indicated. Tumor volume is calculated based on two dimensions,measured using calipers, and is expressed in mm³ according to theformula: V=0.5a×b², where a and b are the long and the short diametersof the tumor, respectively. Data collected from each experimental groupare expressed as mean±SE. Groups of mice are treated with a singleintravenous (i.v.) dose ranging from about 2 to about 8 mg ADC/kg ofwith anti-FcRH5 ADC or control antibody-drug conjugates (ADCs). Tumorsare measured twice a week throughout the experiment. Body weights ofmice are measured once a week for 2 weeks. Mice are euthanized beforetumor volumes reached 3000 mm³ or when tumors show signs of impendingulceration. Negative control include anti-HER2 (HERCEPTIN®(trastuzumab)) based conjugate (SPDB-DM4).

B. Results

1. EJM-FcRH5.LSP.2 Xenografts

At the end of the experiment, tumor size in the control-treated animalsis compared to tumor size in the anti-FcRH5 ADC-treated animals toassess the efficacy of the anti-FcRH5 ADC treatment. Specifically, thenumber of mice out of the total number tested showing Partial Regression(PR; where the tumor volume at any time after administration droppedbelow 50% of the tumor volume measured at day 0), Complete Remission(CR; where the tumor volume at any time after administration dropped to0 mm3) are also determined (NA=Not Applicable); (TI=number of observabletumors at end of study).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

What is claimed is:
 1. A method of determining the presence of FcRH5 ina biological sample suspected of containing FcRH5, said methodcomprising exposing said sample to an isolated monoclonal anti-FcRH5antibody comprising: (a) a light chain variable domain comprising: (i)HVR-L1 comprising the sequence KASQNVGSNVA (SEQ ID NO: 28); (ii) HVR-L2comprising the sequence SASYRYS (SEQ ID NO: 29); and (iii) HVR-L3comprising the sequence QQYKTWT (SEQ ID NO: 30); and (b) a heavy chainvariable domain comprising: (i) HVR-H1 comprising the sequenceGYTFTNYGMN (SEQ ID NO: 37) or GYTFTNYGIN (amino acid residues 26-35 ofSEQ ID NO:22); (ii) HVR-H2 comprising the sequence NTYTGEPTYTDDFKG (SEQID NO: 38); and (iii) HVR-H3 comprising the sequence ARRSIPYYYAMDY (SEQID NO: 39), and determining binding of said antibody to FcRH5 in saidsample wherein binding of said antibody to FcRH5 in said sample isindicative of the presence of FcRH5 in said sample.